Respiratory syncytial virus vaccines expressing protective antigens from promotor-proximal genes

ABSTRACT

Recombinant respiratory syncytial virus (RSV) having the position of genes shifted within the genome or antigenome of the recombinant virus are infectious and attenuated in humans and other mammals. Gene shifted RSV are constructed by insertion, deletion or rearrangement of genes or genome segments within the recombinant genome or antigenome and are useful in vaccine formulations for eliciting an anti-RSV immune response. Also provided are isolated polynucleotide molecules and vectors incorporating a recombinant RSV genome or antigenome wherein a gene or gene segment is shifted to a more promoter-proximal or promoter-distal position within the genome or antigenome compared to a wild type position of the gene in the RSV gene map. Shifting the position of genes in this manner provides for a selected increase or decrease in expression of the gene, depending on the nature and degree of the positional shift. In one embodiment, RSV glycoproteins are upregulated by shifting one or more glycoprotein-encoding genes to a more promoter-proximal position. Genes of interest for manipulation to create gene position-shifted RSV include any of the NS1, NS2, N, P, M, SH, M2(ORF1), M2(ORF2), L, F or G genes or a genome segment that may be part of a gene or extragenic. A variety of additional mutations and nucleotide modifications are provided within the gene position-shifted RSV of the invention to yield desired phenotypic and structural effects.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication No. 60/213,708, filed by Krempl et al. on Jun. 23, 2000.

BACKGROUND OF THE INVENTION

Human respiratory syncytial virus (HRSV) is the leading viral agent ofserious pediatric respiratory tract disease worldwide (Collins, et al.,Fields Virology 2:1313-1352, 1996). RSV outranks all other microbialpathogens as a cause of pneumonia and bronchiolitis in infants under oneyear of age. Virtually all children are infected by two years of age,and reinfection occurs with appreciable frequency in older children andyoung adults (Chanock et al., in Viral Infections of Humans 3rd ed., A.S. Evans, ed., Plenum Press, N.Y., 1989). RSV is responsible for morethan one in five pediatric hospital admissions due to respiratory tractdisease, and in the United States alone causes nearly 100,000hospitalizations and 4,500 deaths yearly. (Heilman, J. Infect Dis161:402-6, 1990). In addition, there is evidence that seriousrespiratory tract infection early in life can initiate or exacerbateasthma (Sigurs, et al., Pediatrics 95:500-5, 1995).

While human RSV usually is thought of in the context of the pediatricpopulation, it also is recognized as an important agent of seriousdisease in the elderly (Falsey, et al., J. Infect. Dis. 172:389-394,1995). Human RSV also causes life-threatening disease in certainimmunocompromised individuals, such as bone marrow transplant recipients(Fouillard, et al., Bone Marrow Transplant 9:97-100, 1992).

For treatment of human RSV, one chemotherapeutic agent, ribavirin, isavailable. However, its efficacy and use is controversial. There arealso licensed products for RSV intervention which are composed of pooleddonor IgG (Groothuis, et al., N Engl J Med 329:1524-30, 1993) or ahumanized RSV-specific monoclonal antibody. These are administered aspassive immunoprophylaxis agents to high risk individuals. While theseproducts are useful, their high cost and other factors, such as lack oflong term effectiveness, make them inappropriate for widespread use.Other disadvantages include the possibility of transmitting blood-borneviruses and the difficulty and expense in preparation and storage.Moreover, the history of the control of infectious diseases, andespecially diseases of viral origin, indicates the primary importance ofvaccines.

Despite decades of investigation to develop effective vaccine agentsagainst RSV, no safe and effective vaccine has yet been achieved toprevent the severe morbidity and significant mortality associated withRSV infection. Failure to develop successful vaccines relates in part tothe fact that small infants have diminished serum and secretory antibodyresponses to RSV antigens. Thus, these individuals suffer more severeinfections from RSV, whereas cumulative immunity appears to protectolder children and adults against more serious impacts of the virus.

The mechanisms of immunity in RSV infection have recently come intofocus. Secretory antibodies appear to be most important in protectingthe upper respiratory tract, whereas high levels of serum antibodies arethought to have a major role in resistance to RSV infection in the lowerrespiratory tract. RSV-specific cytotoxic T cells, another effector armof induced immunity, are also important in resolving an RSV infection.However, while this latter effector can be augmented by priorimmunization to yield increased resistance to virus challenge, theeffect is short-lived. The F and G surface glycoproteins are the twomajor protective antigens of RSV, and are the only two RSV proteinswhich have been shown to induce RSV neutralizing antibodies and longterm resistance to challenge (Collins et al., Fields Virology, Fields etal., eds., 2:1313-1352, Lippincott-Raven, Philadelphia, 1996; Connors etal., J. Virol. 65(3):1634-7, 1991). The third RSV surface protein, SH,did not induce RSV-neutralizing antibodies or significant resistance toRSV challenge.

An obstacle to developing live RSV vaccines is the difficulty inachieving an appropriate balance between attenuation and immunogenicity,partly due to the genetic instability of some attenuated viruses, therelatively poor growth of RSV in cell culture, and the instability ofthe virus particle. In addition the immunity which is induced by naturalinfection is not fully protective against subsequent infection. A numberof factors probably contribute to this, including the relativeinefficiency of the immune system in restricting virus infection on theluminal surface of the respiratory tract, the short-lived nature oflocal mucosal immunity, rapid and extensive virus replication, reducedimmune responses in the young due to immunological immaturity,immunosuppression by transplacentally derived maternal serum antibodies,and certain features of the virus such as a high degree of glycosylationof the G protein. Also, as will be described below, human RSV exists astwo antigenic subgroups A and B, and immunity against one subgroup is ofreduced effectiveness against the other.

Although RSV can reinfect multiple times during life, reinfectionsusually are reduced in severity due to protective immunity induced byprior infection, and thus immunoprophylaxis is feasible. Alive-attenuated RSV vaccine would be administered intranasally toinitiate a mild immunizing infection. This has the advantage ofsimplicity and safety compared to a parenteral route. It also providesdirect stimulation of local respiratory tract immunity, which plays amajor role in resistance to RSV. It also abrogates the immunosuppressiveeffects of RSV-specific maternally-derived serum antibodies, whichtypically are found in the very young. Also, while the parenteraladministration of RSV antigens can sometimes be associated withimmunopathologic complications (Murphy et al., Vaccine 8(5):497-502,1990), this has never been observed with a live virus.

A formalin-inactivated virus vaccine was tested against RSV in themid-1960s, but failed to protect against RSV infection or disease, andin fact exacerbated symptoms during subsequent infection by the virus.(Kim et al., Am. J. Epidemiol., 89:422434, 1969; Chin et al., Am J.Epidemiol., 89:449-463, 1969; Kapikian et al., Am. J. Epidemiol.,89:405421, 1969).

More recently, vaccine development for RSV has focused on attenuated RSVmutants. Friedewald et al., (J. Amer. Med. Assoc. 204:690-694, 1968)reported a cold passaged mutant of RSV (cpRSV) which appeared to besufficiently attenuated to be a candidate vaccine. This mutant exhibiteda slight increased efficiency of growth at 26° C. compared to itswild-type (wt) parental virus, but its replication was neithertemperature sensitive nor significantly cold-adapted. The cold-passagedmutant, however, was attenuated for adults. Although satisfactorilyattenuated and immunogenic for infants and children who had beenpreviously infected with RSV (i.e., seropositive individuals), the cpRSVmutant retained a low level virulence for the upper respiratory tract ofseronegative infants.

Similarly, Gharpure et al., (J. Virol. 3:414-421, 1969) reported theisolation of temperature sensitive RSV (tsRSV) mutants which also werepromising vaccine candidates. One mutant, ts-1, was evaluatedextensively in the laboratory and in volunteers. The mutant producedasymptomatic infection in adult volunteers and conferred resistance tochallenge with wild-type virus 45 days after immunization. Again, whileseropositive infants and children underwent asymptomatic infection,seronegative infants developed signs of rhinitis and other mildsymptoms. Furthermore, instability of the ts phenotype was detected.Although virus exhibiting a partial or complete loss of temperaturesensitivity represented a small proportion of virus recoverable fromvaccinees, it was not associated with signs of disease other than mildrhinitis.

These and other studies revealed that certain cold-passaged andtemperature sensitive RSV strains were underattenuated and caused mildsymptoms of disease in some vaccinees, particularly seronegativeinfants, while others were overattenuated and failed to replicatesufficiently to elicit a protective immune response, (Wright et al.,Infect. Immun., 37:397-400, 1982). Moreover, genetic instability ofcandidate vaccine mutants has resulted in loss of their temperaturesensitive phenotype, further hindering development of effective RSVvaccines. See generally, (Hodes et al., Proc. Soc. Exp. Biol. Med.145:1158-1164, 1974; McIntosh et al., Pediatr. Res. 8:689-696, 1974; andBelshe et al., J. Med. Virol., 3:101-110, 1978).

As an alternative to live-attenuated RSV vaccines, investigators havealso tested subunit vaccine candidates using purified RSV envelopeglycoproteins. The glycoproteins induced resistance to RS virusinfection in the lungs of cotton rats, (Walsh et al., J. Infect. Dis.155:1198-1204, 1987), but the antibodies had very weak neutralizingactivity and immunization of rodents with purified subunit vaccine ledto disease potentiation (Murphy et al., Vaccine 8:497-502, 1990).

Recombinant vaccinia virus vaccines which express the F or G envelopeglycoprotein have also been explored. These recombinants express RSVglycoproteins which are indistinguishable from the authentic viralcounterpart, and rodents infected intradermally with vaccinia-RSV F andG recombinants developed high levels of specific antibodies thatneutralized viral infectivity. Indeed, infection of cotton rats withvaccinia-F recombinants stimulated almost complete resistance toreplication of RSV in the lower respiratory tract and significantresistance in the upper tract. (Olmsted et al., Proc. Natl. Acad. Sci.USA 83:7462-7466, 1986). However, immunization of chimpanzees withvaccinia-F and -G recombinant provided almost no protection against RSVchallenge in the upper respiratory tract (Collins et al., Vaccine8:164-168, 1990) and inconsistent protection in the lower respiratorytract (Crowe et al., Vaccine 11: 1395-1404, 1993).

Despite these various efforts to develop an effective RSV vaccine, nolicensed vaccine has yet been approved for RSV. The unfulfilled promisesof prior approaches underscores a need for new strategies to develop RSVvaccines, and in particular methods for manipulating recombinant RSV toincorporate genetic changes that yield new phenotypic properties inviable, attenuated RSV recombinants. However, manipulation of thegenomic RNA of RSV and other non-segmented negative-sense RNA viruseshas heretofore proven difficult. Major obstacles in this regard includenon-infectivity of naked genomic RNA of these viruses, poor viral growthin tissue culture, lengthy replication cycles, virion instability, acomplex genome, and a refractory organization of gene products.

Recombinant DNA technology has made it possible to recover infectiousnon-segmented negative-stranded RNA viruses from cDNA, to geneticallymanipulate viral clones to construct novel vaccine candidates, and torapidly evaluate their level of attenuation and phenotypic stability(for reviews, see Conzelmann, J. Gen. Virol. 77:381-89, 1996; Palese etal., Proc. Natl. Acad. Sci. U.S.A. 93:11354-58, 1996). In this context,recombinant rescue has been reported for infectious respiratorysyncytial virus (RSV), parainfluenza virus (PIV), rabies virus (RaV),vesicular stomatitis virus (VSV), measles virus (MeV), rinderpest virusand Sendai virus (SeV) from cDNA-encoded antigenomic RNA in the presenceof essential viral proteins (see, e.g., Garcin et al., EMBO J.14:6087-6094, 1995; Lawson et al., Proc. Natl. Acad. Sci. U.S.A.92:4477-81, 1995; Radecke et al., EMBO J. 14:5773-5784, 1995; Schnell etal., EMBO J. 13:4195-203, 1994; Whelan et al., Proc. Natl. Acad. Sci.U.S.A. 92:8388-92, 1995; Hoffman et al., J. Virol. 71:4272-4277, 1997;Pecters et al., J. Virol. 73:5001-5009, 1999; Kato et al., Genes toCells 1:569-579, 1996; Roberts et al., Virology 247(1), 1-6, 1998; Baronet al., J. Virol. 71:1265-1271, 1997; International Publication No. WO97/06270; U.S. Provisional Patent Application No. 60/007,083, filed Sep.27, 1995; U.S. patent application Ser. No. 08/720,132, filed Sep. 27,1996; U.S. Provisional Patent Application No. 60/021,773, filed Jul. 15,1996; U.S. Provisional Patent Application No. 60/046,141, filed May 9,1997; U.S. Provisional Patent Application No. 60/047,634, filed May 23,1997; U.S. Pat. No. 5,993,824, issued Nov. 30, 1999 (corresponding toInternational Publication No. WO 98/02530); U.S. patent application Ser.No. 09/291,894, filed by Collins et al. on Apr. 13, 1999; U.S.Provisional Patent Application No. 60/129,006, filed by Murphy et al. onApr. 13, 1999; Collins, et al., Proc Nat. Acad. Sci. U.S.A.92:11563-11567, 1995; Bukreyev, et al., J Virol 70:663441, 1996, Juhaszet al., J. Virol. 71(8):5814-5819, 1997; Durbin et al., Virology235:323-332, 1997; He et al. Virology 237:249-260, 1997; Baron et al. J.Virol. 71:1265-1271, 1997; Whitehead et al., Virology 247(2):232-9,1998a; Buchholz et al. J. Virol. 73:251-9, 1999; Whitehead et al., J.Virol. 72(5):4467-4471, 1998b; Jin et al. Virology 251:206-214, 1998;and Whitehead et al., J. Virol. 73:(4)3438-3442, 1999, and Bukreyev, etal., Proc Nat Acad Sci USA 96:2367-72, 1999, each incorporated herein byreference in its entirety for all purposes).

Based on the foregoing developments, it is now possible to recoverinfectious RSV from cDNA and to design and implement various geneticmanipulations to RSV clones to construct novel vaccine candidates.Thereafter, the level of attenuation and phenotypic stability, amongother desired phenotypic characteristics, can be evaluated and adjusted.The challenge which thus remains is to develop a broad and diverse menuof genetic manipulations that can be employed, alone or in combinationwith other types of genetic manipulations, to construct infectious,attenuated RSV clones that are useful for broad vaccine use. In thiscontext, an urgent need remains in the art for additional tools andmethods that will allow engineering of safe and effective vaccines toalleviate the serious health problems attributable to RSV. Surprisingly,the present invention fulfills this need by providing additional toolsfor constructing infectious, attenuated RSV vaccine candidates.

SUMMARY OF THE INVENTION

The present invention provides recombinant respiratory syncytial viruses(RSVs) which are modified by shifting a relative gene order or spatialposition of one or more genes or genome segments within a recombinantRSV genome or antigenome—to generate a recombinant vaccine virus that isinfectious, attenuated and immunogenic in humans and other mammals. Therecombinant RSVs of the invention typically comprise a majornucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), a largepolymerase protein (L), a RNA polymerase elongation factor, and apartial or complete recombinant RSV genome or antigenome having one ormore positionally shifted RSV genes or genome segments within therecombinant genome or antigenome. In certain aspects of the invention,the recombinant RSV features one or more positionally shifted genes orgenome segments that may be shifted to a more promoter-proximal orpromoter-distal position by insertion, deletion, or rearrangement of oneor more displacement polynucleotides within the partial or completerecombinant RSV genome or antigenome. Displacement polynucleotides maybe inserted or rearranged into a non-coding region (NCR) of therecombinant genome or antigenome, or may be incorporated in therecombinant RSV genome or antigenome as a separate gene unit (GU).

In exemplary embodiments of the invention, isolated infectiousrecombinant RSV are constructed by addition, deletion, or rearrangementof one or more displacement polynucleotides that may be selected fromone or more RSV gene(s) or genome segment(s) selected from RSV NS1, NS2,N, P, M, SH, M2(ORF1), M2(ORF2), L, F and G genes and genome segmentsand leader, trailer and intergenic regions of the RSV genome andsegments thereof. In more detailed embodiments, polynucleotide inserts,and deleted or rearranged elements within the recombinant RSV genome orantigenome are selected, from one or more bovine RSV (BRSV) or human RSV(HRSV) gene(s) or genome segment(s) selected from RSV NS1, NS2, N, P, M,SH, M2(ORF1), M2(ORF2), L, F and G gene(s) or genome segment(s) andleader, trailer and intergenic regions of the RSV genome or segmentsthereof.

In certain aspects of the invention, displacement polynucleotides aredeleted to form the recombinant RSV genome or antigenome. Deletion of adisplacement polynucleotide in this manner causes a positional shift ofone or more “shifted” RSV genes or genome segments within therecombinant genome or antigenome to a more promoter-proximal positionrelative to a position of the shifted gene(s) or genome segment(s)within a wild type RSV (e.g., HRSV A2 or BRSV kansas strain) genome orantigenome. Exemplary displacement polynucleotides that may be deletedin this manner to form the recombinant RSV genome or antigenome may beselected from one or more RSV NS1, NS2, SH, M2(ORF2), or G gene(s) orgenome segment(s) thereof.

In more detailed embodiments of the invention, a displacementpolynucleotide comprising a RSV NS1 gene is deleted to form therecombinant RSV genome or antigenome. Alternatively, a displacementpolynucleotide comprising a RSV NS2 gene may be deleted to form therecombinant RSV genome or antigenome. Alternatively, a displacementpolynucleotide comprising a RSV SH gene may be deleted to form therecombinant RSV genome or antigenome. Alternatively, a displacementpolynucleotide comprising RSV M2(ORF2) can be deleted to form therecombinant RSV genome or antigenome. Alternatively, a displacementpolynucleotide comprising a RSV G gene may be deleted to form therecombinant RSV genome or antigenome or antigenome.

In yet additional embodiments, multiple displacement polynucleotidescomprising RSV genes or genome segments may be deleted. For example, RSVF and G genes may both be deleted to form the recombinant RSV genome orantigenome or antigenome. Alternatively, the RSV NS1 and NS2 genes mayboth be deleted to form the recombinant RSV genome or antigenome orantigenome. Alternatively, the RSV SH and NS2 genes may both be deletedto form the recombinant RSV genome or antigenome or antigenome.Alternatively, the RSV SH, NS1 and NS2 genes can all be deleted to formthe recombinant RSV genome or antigenome or antigenome.

In different embodiments of the invention, isolated infectiousrecombinant RSV are provided wherein one or more displacementpolynucleotides is/are added, substituted, or rearranged within therecombinant RSV genome or antigenome to cause a positional shift of oneor more shifted RSV gene(s) or genome segment(s). Among thesemodifications, gene and genome segment insertions and rearrangements mayintroduce or rearrange the subject genes or genome segments to a morepromoter-proximal or promoter-distal position relative to a respectiveposition of each subject (inserted or rearranged) gene or genome segmentwithin a corresponding (e.g., bovine or human) wild type RSV genome orantigenome. Displacement polynucleotides which may be added,substituted, or rearranged within the recombinant RSV genome orantigenome can be selected from one or more of the RSV NS1, NS2, SH,M2(ORF2), F, and/or G gene(s) or genome segment(s) thereof.

In more detailed embodiments, displacement polynucleotides are selectedfor insertion or rearrangement within the recombinant RSV genome orantigenome which comprises one or more RSV genes or genome segments thatencoded one or more RSV glycoproteins or immunogenic domains or epitopesof RSV glycoproteins. In exemplary embodiments, these displacementpolynucleotides are selected from genes or genome segments encoding RSVF, G, and/or SH glycoproteins or immunogenic domains or epitopesthereof. For example, one or more RSV glycoprotein gene(s) selected fromF, G and SH may be added, substituted or rearranged within therecombinant RSV genome or antigenome to a position that is morepromoter-proximal or promoter-distal compared to the wild type geneorder position of the gene(s).

In exemplary embodiments, the RSV glycoprotein gene G is rearrangedwithin the recombinant RSV genome or antigenome to a gene order positionthat is more promoter-proximal compared to the wild type gene orderposition of G. In more detailed aspects, the RSV glycoprotein gene G isshifted to gene order position 1 within said recombinant RSV genome orantigenome. In other exemplary embodiments, the RSV glycoprotein gene Fis rearranged within the recombinant RSV genome or antigenome to a morepromoter-proximal position, for example by shifting the F gene to geneorder position 1 within the recombinant genome or antigenome. In yetadditional exemplary embodiments, both RSV glycoprotein genes G and Fare rearranged within the recombinant RSV genome or antigenome to geneorder positions that are more promoter-proximal compared to theirrespective wild type gene order positions. In more detailed aspects, theRSV glycoprotein gene G is shifted to gene order position 1 and the RSVglycoprotein gene F is shifted to gene order position 2.

In yet additional constructs featuring glycoprotein gene shifts,recombinant RSV are produced having one or more RSV glycoprotein gene(s)selected from F, G and SH, or a genome segment thereof, added,substituted or rearranged within the recombinant RSV genome orantigenome, wherein one or more RSV NS1, NS2, SH, M2(ORF2), or G gene(s)or genome segment(s) thereof is/are deleted. Thus, a gene or genomesegment of RSV F, G, or SH may be added, substituted or rearranged in abackground wherein a displacement polynucleotide comprising a RSV NS1gene is deleted to form the recombinant RSV genome or antigenome.Alternatively, a gene or genome segment of RSV F, G, or SH may be added,substituted or rearranged in a background wherein a displacementpolynucleotide comprising a RSV NS2 gene is deleted to form therecombinant RSV genome or antigenome. Alternatively, a gene or genomesegment of RSV F, G, or SH may be added, substituted or rearranged in abackground wherein a displacement polynucleotide comprising a RSV SHgene is deleted to form the recombinant RSV genome or antigenome.

In one example below, the RSV glycoprotein gene G is rearranged within arecombinant RSV genome or antigenome having an SH gene deletion to agene order position that is more promoter-proximal compared to the wildtype gene order position of G. In more detailed aspects, the RSVglycoprotein gene G is shifted to gene order position 1 within therecombinant RSV genome or antigenome, as exemplified by the recombinantvaccine candidate G1/ΔSH. In another example, the RSV glycoprotein geneF is rearranged within a recombinant RSV genome or antigenome having anSH gene deletion to a more promoter-proximal proximal position. In moredetailed aspects, the F gene is shifted to gene order position 1, asexemplified by the recombinant F1ΔSH. In yet another example below, bothRSV glycoprotein genes G and F are rearranged within a ΔSH recombinantRSV genome or antigenome to gene order positions that are morepromoter-proximal compared to the wild type gene order positions of Gand F. In more detailed aspects, the RSV glycoprotein gene G is shiftedto gene order position 1 and the RSV glycoprotein gene F is shifted togene order position 1 within the recombinant RSV genome or antigenome,as exemplified by the recombinant G1F2/ΔSH.

Yet additional examples of gene position-shifted RSV are providedfeaturing shifts of glycoprotein gene(s) selected from F, G and SH,which are produced within a recombinant RSV genome or antigenome havingmultiple genes or genome segments selected from RSV NS1, NS2, SH,M2(ORF2), and G gene(s) or genome segment(s) deleted. In one example,the RSV SH and NS2 genes are both deleted to form the recombinant RSVgenome or antigenome or antigenome, and one or both RSV glycoproteingenes G and F are rearranged within the recombinant RSV genome to morepromoter-proximal gene order positions. In more detailed aspects, G isshifted to gene order position 1 and F is shifted to gene order position2, as exemplified by the recombinant G1F2/ΔNS2ΔSH. In another example,all of the RSV SH, NS1 and NS2 genes are deleted to form the recombinantRSV genome or antigenome or antigenome, and one or both RSV glycoproteingenes G and F are rearranged within the recombinant RSV genome orantigenome to more promoter-proximal positions, as exemplified by therecombinant vaccine candidate G1F2/ΔNS2ΔNS2ΔSH.

In yet additional aspects of the invention, gene position-shifted RSVare combined with or incorporated within human-bovine chimeric RSV.Within these aspects, the recombinant genome or antigenome comprises apartial or complete human RSV (HRSV) or bovine RSV (BRSV) backgroundgenome or antigenome combined with one or more heterologous gene(s) orgenome segment(s) from a different RSV to for a human-bovine chimericRSV genome or antigenome. The heterologous gene or genome segment of thedifferent, HRSV or BRSV may be added or substituted at a position thatis more promoter-proximal or promoter-distal compared to a wild typegene order position of a counterpart gene or genome segment within thepartial or complete HRSV or BRSV background genome or antigenome. In onesuch example provided herein, both human RSV glycoprotein genes G and Fare substituted at gene order positions 1 and 2, respectively, toreplace counterpart G and F glycoprotein genes deleted at wild typepositions 7 and 8, respectively in a partial bovine RSV backgroundgenome or antigenome, as exemplified by the recombinant virusrBRSV/A2-G1F2. In other embodiments, one or more human RSVenvelope-associated genes selected from F, G, SH, and M is/are added orsubstituted within a partial or complete bovine RSV background genome orantigenome. In more detailed aspects, one or more human RSVenvelope-associated genes selected from F, G, SH, and M is/are added orsubstituted within a partial bovine RSV background genome or antigenomein which one or more envelope-associated genes selected from F, G, SH,and M is/are deleted. In one example described below, human RSVenvelope-associated genes F, G, and M are added within a partial bovineRSV background genome or antigenome in which all of theenvelope-associated genes F, G, SH, and M are deleted, as exemplified bythe recombinant virus rBRSV/A2-MGF.

In another alternate embodiment of the invention, isolated infectiousrecombinant RSV are provided in which the RSV M2(ORF1) is shifted to amore promoter-proximal position within the recombinant RSV genome orantigenome. The result of this gene shift is to upregulate transcriptionof the recombinant virus.

In additional aspects of the invention, attenuated, geneposition-shifted RSV are produced in which the recombinant genome orantigenome is further modified by introducing one or more attenuatingmutations specifying an attenuating phenotype in the resultant virus orsubviral particle. These attenuating mutations may be generated de novoand tested for attenuating effects according to a rational designmutagenesis strategy. Alternatively, the attenuating mutations may beidentified in existing biologically derived mutant RSV and thereafterincorporated into a gene position-shifted RSV of the invention.

In combination with the gene positional changes introduced inrecombinant RSV of the invention, it is often desirable to adjust theattenuation phenotype by introducing additional mutations that increaseor decrease attenuation of the chimeric virus. Thus, candidate vaccinestrains can be further attenuated by incorporation of at least one, andpreferably two or more different attenuating mutations, for examplemutations identified from a panel of known, biologically derived mutantRSV strains. Preferred human mutant RSV strains are cold passaged (cp)and/or temperature sensitive (ts) mutants, for example the mutantsdesignated “cpts RSV 248 (ATCC VR 2450), cpts RSV 248/404 (ATCC VR2454), cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452),cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSVB-1 cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCC VR 2579)” (eachdeposited under the terms of the Budapest Treaty with the American TypeCulture Collection (ATCC) of 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., and granted the above identified accession numbers).From this exemplary panel of biologically derived mutants, a large“menu” of attenuating mutations are provided which can each be combinedwith any other mutation(s) within the panel for calibrating the level ofattenuation in the recombinant, human-bovine chimeric RSV for vaccineuse. Additional mutations may be derived from RSV having non-ts andnon-cp attenuating mutations as identified, e.g., in small plaque (sp),cold-adapted (ca) or host-range restricted (hr) mutant strains. Themutations may be incorporated in either a human or bovine antigenomicsequence, and attenuating mutations identified in a human, bovine orother RSV mutant may be transferred to the heterologous RSV recipient(e.g., bovine or human RSV, respectively) by mapping the mutation to thecorresponding, homologous site in the recipient genome and mutating thenative sequence in the recipient to the mutant genotype (either by anidentical or conservative mutation), as described in U.S. ProvisionalPatent Application No. 60/129,006, filed by Murphy et al. on Apr. 13,1999, incorporated herein by reference.

Thus, in more detailed embodiments of the invention, geneposition-shifted RSV are provided wherein the recombinant genome orantigenome incorporates at least one and up to a full complement ofattenuating mutations present within a panel of mutant human RSVstrains, said panel comprising cpts RSV 248 (ATCC VR 2450), cpts RSV248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530(ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030(ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCCVR 2579). In certain embodiments, the recombinant genome or antigenomeincorporates attenuating mutations adopted from different mutant RSVstrains.

Gene position-shifted RSV designed and selected for vaccine use oftenhave at least two and sometimes three or more attenuating mutations toachieve a satisfactory level of attenuation for broad clinical use. Inone embodiment, at least one attenuating mutation occurs in the RSVpolymerase gene L (either in the donor or recipient gene) and involvesone or more nucleotide substitution(s) specifying an amino acid changein the polymerase protein specifying an attenuation phenotype which mayor may not involve a temperature-sensitive (ts) phenotype. Geneposition-shifted RSV of the invention may incorporate an attenuatingmutation in any additional RSV gene besides L, e.g., in the M2 gene.However, preferred human-bovine chimeric RSV in this context incorporateone or more nucleotide substitutions in the large polymerase gene Lresulting in an amino acid change at amino acid Asn43, Cys319, Phe 521,Gln831, Met1169, Tyr1321 and/or His 1690 in the RSV polymerase gene L,as exemplified by the changes, Ile for Asn43, Leu for Phe521, Leu forGln831, Val for Met1169, and Asn for Tyr1321. Other alternative aminoacid changes, particularly conservative changes with respect toidentified mutant residues, at these positions can of course be made toyield a similar effect as the identified, mutant substitution.Additional desired mutations for incorporation into human-bovinechimeric RSV of the invention include attenuating mutations specifyingan amino acid substitution at Val267 in the RSV N gene, Glu218 and/orThr523 in the RSV F gene, and a nucleotide substitution in thegene-start sequence of gene M2. Any combination of one or moreattenuating mutations identified herein, up to and including a fullcomplement of these mutations, may be incorporated in human-bovinechimeric RSV to yield a suitably attenuated recombinant virus for use inselected populations or broad populations of vaccine recipients.

In other more detailed embodiments of the invention, geneposition-shifted RSV are provided wherein the recombinant genome orantigenome incorporates at least one and up to a full complement ofattenuating mutations specifying an amino acid substitution at Val267 inthe RSV N gene, Glu218 and/or Thr523 in the RSV F gene, Asn43, Cys319,Phe 521, Gln831, Met1169, Tyr1321 and/or His 1690 in the RSV polymerasegene L, and a nucleotide substitution in the gene-start sequence of geneM2. In certain aspects, the recombinant genome or antigenomeincorporates at least two, commonly three, four or five, and sometimes afull complement comprising all of these attenuating mutations. Often, atleast one attenuating mutation is stabilized by multiple nucleotidechanges in a codon specifying the mutation.

Attenuating mutations for incorporation in human-bovine chimeric RSV ofthe invention may be selected in coding portions of a donor or recipientRSV gene or in non-coding regions such as a cis-regulatory sequence.Exemplary non-coding mutations include single or multiple base changesin a gene start sequence, as exemplified by a single or multiple basesubstitution in the M2 gene start sequence at nucleotide 7605(nucleotide 7606 in recombinant sequence).

Infectious RSV according to the invention can incorporate heterologous,coding or non-coding nucleotide sequences from any heterologous RSV orRSV-like virus, e.g., human, bovine, murine pneumonia virus of mice), oravian (turkey rhinotracheitis virus) pneumovirus, or from anotherenveloped virus, e.g., parainfluenza virus (PIV). Exemplary heterologoussequences include RSV sequences from one human RSV strain combined withsequences from a different human RSV strain, or RSV sequences from ahuman RSV strain combined with sequences from a bovine RSV strain. Geneposition-shifted RSV of the invention may incorporate sequences from twoor more wild-type or mutant RSV strains, for example mutant stainsselected from cpts RSV 248, cpts 248/404, cpts 248/955, cpts RSV 530,cpts 530/1009, or cpts 530/1030. Alternatively, chimeric RSV mayincorporate sequences from two or more, wild-type or mutant human RSVsubgroups, for example a combination of human RSV subgroup A andsubgroup B sequences. In yet additional aspects, one or more human RSVcoding or non-coding polynucleotides are substituted with a counterpartsequence from a heterologous RSV or non-RSV virus, alone or incombination with one or more selected attenuating mutations, e.g., cpand/or ts mutations, to yield novel attenuated vaccine strains.

Mutations incorporated within gene position-shifted RSV cDNAs, vectorsand viral particles of the invention can be introduced individually orin combination into a full-length RSV cDNA, and the phenotypes ofrescued virus containing the introduced mutations can be readilydetermined. In exemplary embodiments, amino acid changes displayed byattenuated, biologically-derived viruses versus a wild-type RSV, forexample changes exhibited by cpRSV or tsRSV, are incorporated incombination within a gene position-shifted RSV to yield a desired levelof attenuation.

In additional aspects of the invention, gene position-shifted RSV can bereadily designed as “vectors” to incorporate antigenic determinants fromdifferent pathogens, including more than one RSV strain or group (e.g.,both human RSV A and RSV B subgroups), human parainfluenza virus (HPIV)including HPIV3, HPIV2 and HPIV1, measles virus and other pathogens(see, e.g., U.S. Provisional Patent Application Ser. No. 60/170,195;U.S. patent application Ser. No. 09/458,813; and U.S. patent applicationSer. No. 09/459,062, each incorporated herein by reference). Withinvarious embodiments, the recombinant genome or antigenome comprises apartial or complete RSV “vector genome or antigenome” combined with oneor more heterologous genes or genome segments encoding one or moreantigenic determinants of one or more heterologous pathogens. Theheterologous pathogen may be a heterologous RSV (i.e., a RSV of adifferent strain or subgroup), and the heterologous gene or genomesegment may encode a RSV NS1, NS2, N, P, M, SH, M2(ORF1), M2(ORF2), L, For G protein or fragment (e.g., a immunogenic domain or epitope)thereof. For example, the vector genome or antigenome may be a partialor complete RSV A genome or antigenome and the heterologous gene(s) orgenome segment(s) may encode antigenic determinant(s) of a RSV Bsubgroup virus.

In alternative embodiments, the gene position-shifted RSV vector genomeor antigenome is a partial or complete BRSV genome or antigenome and theheterologous gene(s) or genome segment(s) encoding the antigenicdeterminant(s) is/are of one or more HRSV(s). For example, the partialor complete BRSV genome or antigenome may incorporate one or moregene(s) or genome segment(s) encoding one or more HRSV glycoproteingenes selected from F, G and SH, or one or more genome segment(s)encoding cytoplasmic domain, transmembrane domain, ectodomain orimmunogenic epitope portion(s) of F, G, and/or SH of HRSV.

As noted above, gene position-shifted RSV designed as vectors forcarrying heterologous antigenic determinants may incorporate one or moreantigenic determinants of a non-RSV pathogen, such as a humanparainfluenza virus (HPIV). In one exemplary embodiment, one or moreHPIV1, HPIV2, or HPIV3 gene(s) or genome segment(s) encoding one or moreHN and/or F glycoprotein(s) or antigenic domain(s), fragment(s) orepitope(s) thereof is/are added to or incorporated within the partial orcomplete HRSV vector genome or antigenome. In more detailed embodiments,a transcription unit comprising an open reading frame (ORF) of an HPIV1,BPIV2, or HPIV3 HN or F gene is added to or incorporated within therecombinant vector genome or antigenome.

In yet additional alternate embodiments, the vector genome or antigenomecomprises a partial or complete HRSV or BRSV genome or antigenome andthe heterologous pathogen is selected from measles virus, subgroup A andsubgroup B respiratory syncytial viruses, mumps virus, human papillomaviruses, type 1 and type 2 human immunodeficiency viruses, herpessimplex viruses, cytomegalovirus, rabies virus, Epstein Barr virus,filoviruses, bunyaviruses, flaviviruses, alphaviruses and influenzaviruses. Based on this exemplary list of candidate pathogens, theselected heterologous antigenic determinant(s) may be selected frommeasles virus HA and F proteins, subgroup A or subgroup B respiratorysyncytial virus F, G, SH and M2 proteins, mumps virus HN and F proteins,human papilloma virus L1 protein, type 1 or type 2 humanimmunodeficiency virus gp160 protein, herpes simplex virus andcytomegalovirus gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, and gM proteins,rabies virus G protein, Epstein Barr Virus gp350 protein; filovirus Gprotein, bunyavirus G protein, Flavivirus E and NS1 proteins, andalphavirus E protein, and antigenic domains, fragments and epitopesthereof. In one embodiment, the heterologous pathogen is measles virusand the heterologous antigenic determinant(s) is/are selected from themeasles virus HA and F proteins and antigenic domains, fragments andepitopes thereof. To achieve such a chimeric construct, a transcriptionunit comprising an open reading frame (ORF) of a measles virus HA genemay be added to or incorporated within a HRSV vector genome orantigenome.

The present invention thus provides gene position-shifted RSV clones,polynucleotide expression constructs (also referred to as vectors) andparticles which can incorporate multiple, phenotype-specific mutationsintroduced in selected combinations into the gene position-shifted RSVgenome or antigenome to produce an attenuated, infectious virus orsubviral particle. This process coupled with routine phenotypicevaluation provides gene position-shifted RSV having such desiredcharacteristics as attenuation, temperature sensitivity, alteredimmunogenicity, cold-adaptation, small plaque size, host rangerestriction, etc. Mutations thus identified are compiled into a “menu”and introduced in various combinations to calibrate a vaccine virus to aselected level of attenuation, immunogenicity and stability.

In yet additional aspects of the invention, gene position-shifted RSV,with or without attenuating mutations, are constructed to have anucleotide modification to yield a desired phenotypic, structural, orfunctional change. Typically, the selected nucleotide modification willspecify a phenotypic change, for example a change in growthcharacteristics, attenuation, temperature-sensitivity, cold-adaptation,plaque size, host range restriction, or immunogenicity. Structuralchanges in this context include introduction or ablation of restrictionsites into RSV encoding cDNAs for ease of manipulation andidentification.

In certain embodiments, nucleotide changes within gene position-shiftedRSV include modification of a viral gene by deletion of the gene orablation of its expression. Target genes for mutation in this contextinclude the attachment (G) protein, fusion (F) protein, smallhydrophobic (SH), RNA binding protein (N), phosphoprotein (P), the largepolymerase protein (L), the transcription elongation factor (M2 ORF1),the RNA regulatory factor M2 ORF2, the matrix (M) protein, and twononstructural proteins, NS1 and NS2. Each of these proteins can beselectively deleted, substituted or rearranged, in whole or in part,alone or in combination with other desired modifications, to achievenovel chimeric RSV recombinants.

In one aspect of the invention, an SH, NS1, NS2, G or M2-2 gene ismodified in the gene position-shifted RSV. For example, each of thesegenes may be deleted or its expression ablated (e.g., by introduction ofa stop codon) to alter the phenotype of the resultant recombinant RSVclone to improve growth, attenuation, immunogenicity or other desiredphenotypic characteristics. For example, deletion of the SH gene in therecombinant genome or antigenome will yield a RSV having novelphenotypic characteristics such as enhanced growth in vitro and/orattenuation in vivo. In a related aspect, an SH gene deletion, ordeletion of another selected non-essential gene or genome segment suchas a NS1, NS2, G or M2-2 gene deletion is constructed in a geneposition-shifted RSV, alone or in combination with one or more differentmutations specifying an attenuated phenotype, e.g., a point mutationadopted directly (or in modified form, e.g., by introducing multiplenucleotide changes in a codon specifying the mutation) from abiologically derived attenuated RSV mutant. For example, the SH, NS1,NS2, G or M2-2 gene may be deleted in combination with one or more cpand/or ts mutations adopted from cpts248/404, cpts530/1009, cpts530/1030or another selected mutant RSV strain, to yield a gene position-shiftedRSV having increased yield of virus, enhanced attenuation, improvedimmunogenicity and genetic resistance to reversion from an attenuatedphenotype due to the combined effects of the different mutations.

Alternative nucleotide modifications can include a deletion, insertion,addition or rearrangement of a cis-acting regulatory sequence for aselected gene in the gene position-shifted RSV. In one example, acis-acting regulatory sequence of one RSV gene is changed to correspondto a heterologous regulatory sequence, which may be a counterpartcis-acting regulatory sequence of the same gene in a different RSV or acis-acting regulatory sequence of a different RSV gene. For example, agene end signal may be modified by conversion or substitution to a geneend signal of a different gene in the same RSV strain. In otherembodiments, the nucleotide modification may comprise an insertion,deletion, substitution, or rearrangement of a translational start sitewithin the chimeric genome or antigenome, e.g., to ablate an alternativetranslational start site for a selected form of a protein. In oneexample, the translational start site for a secreted form of the RSV Gprotein is ablated to modify expression of this form of the G proteinand thereby produce desired in vivo effects.

In addition, a variety of other genetic alterations can be produced in agene position-shifted RSV genome or antigenome, alone or together withone or more attenuating mutations adopted from a biologically derivedmutant RSV. For example, genes or genome segments from non-RSV sourcesmay be inserted in whole or in part. Nontranslated gene sequences can beremoved, e.g., to increase capacity for inserting foreign sequences. Inyet additional aspects, polynucleotide molecules or vectors encoding thegene position-shifted RSV genome or antigenome can be modified to encodenon-RSV sequences, e.g., a cytokine, a T-helper epitope, a restrictionsite marker, or a protein of a microbial pathogen (e.g., virus,bacterium or fungus) capable of eliciting a protective immune responsein an intended host. Different or additional modifications in the geneposition-shifted RSV antigenome can be made to facilitate manipulations,such as the insertion of unique restriction sites in various intergenicregions (e.g., a unique StuI site between the G and F genes) orelsewhere.

All of the foregoing modifications within the gene position-shifted RSVgenome or antigenome, including nucleotide insertions, rearrangements,deletions or substitutions yielding point mutations, site-specificnucleotide changes, and changes involving entire genes or genomesegments, may be made to either a partial or complete RSV genome orantigenome, or within a heterologous donor gene or genome segment orrecipient, background genome or antigenome in a chimeric RSV. In eachcase, these alterations will preferably specify one or more phenotypicchange(s) in the resulting recombinant RSV, such as a phenotypic changethat results in attenuation, temperature-sensitivity, cold-adaptation,small plaque size, host range restriction, alteration in geneexpression, or a change in an immunogenic epitope.

In another aspect of the invention, compositions (e.g., isolatedpolynucleotides and vectors incorporating an RSV-encoding cDNA) andmethods are provided for producing an isolated infectious geneposition-shifted RSV. Using these compositions and methods, infectiousgene position-shifted RSV particles or subviral particles are generatedfrom a recombinant RSV genome or antigenome coexpressed with anucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), a large (L)polymerase protein, and an RNA polymerase elongation factor. In relatedaspects of the invention, compositions and methods are provided forintroducing the aforementioned structural and phenotypic changes into arecombinant gene position-shifted RSV to yield infectious, attenuatedvaccine viruses.

In one embodiment, an expression vector is provided which comprises anisolated polynucleotide molecule encoding a gene position-shifted RSVgenome or antigenome. Also provided is the same or different expressionvector comprising one or more isolated polynucleotide molecules encodingN, P, L and RNA polymerase elongation factor proteins. These proteinsalso can be expressed directly from the genome or antigenome cDNA. Thevector(s) is/are preferably expressed or coexpressed in a cell orcell-free lysate, thereby producing an infectious gene position-shiftedRSV particle or subviral particle.

The RSV genome or antigenome and the N, P, L and RNA polymeraseelongation factor (preferably the product of the M2(ORF1) of RSV)proteins can be coexpressed by the same or different expression vectors.In some instances the N, P, L and RNA polymerase elongation factorproteins are each encoded on different expression vectors. Thepolynucleotide molecule encoding the gene position-shifted RSV genome orantigenome is operably joined to these control sequences to allowproduction of infectious virus or viral particles therefrom. Inalternative aspects of the invention, the gene position-shifted RSVgenome or antigenome can include sequences from multiple human RSVstrains or subgroups (A and B), as well as other non-human (e.g.,murine) RSV sequences. In other alternate aspects, the geneposition-shifted RSV genome or antigenome can incorporate non-RSVsequences, for example a polynucleotide containing sequences from humanand bovine RSV operably joined with a nucleotide or polynucleotideencoding a point mutation, protein, protein domain or immunogenicepitope of PIV or another negative stranded RNA virus.

The above methods and compositions for producing gene position-shiftedRSV yield infectious viral or subviral particles, or derivativesthereof. An infectious virus is comparable to the authentic RSV virusparticle and is infectious as is. It can directly infect fresh cells. Aninfectious subviral particle typically is a subcomponent of the virusparticle which can initiate an infection under appropriate conditions.For example, a nucleocapsid containing the genomic or antigenomic RNAand the N, P, L and M2(ORF1) proteins is an example of a subviralparticle which can initiate an infection if introduced into thecytoplasm of cells. Subviral particles provided within the inventioninclude viral particles which lack one or more protein(s), proteinsegment(s), or other viral component(s) not essential for infectivity.

In other embodiments the invention provides a cell or cell-free lysatecontaining an expression vector which comprises an isolatedpolynucleotide molecule encoding a gene position-shifted RSV genome orantigenome as described above, and an expression vector (the same ordifferent vector) which comprises one or more isolated polynucleotidemolecules encoding the N, P, L and RNA polymerase elongation factorproteins of RSV. One or more of these proteins also can be expressedfrom the genome or antigenome cDNA. Upon expression the genome orantigenome and N, P, L, and RNA polymerase elongation factor proteinscombine to produce an infectious gene position-shifted RSV viral orsubviral particle.

Attenuated gene position-shifted RSV of the invention is capable ofeliciting a protective immune response in an infected human host, yet issufficiently attenuated so as to not cause unacceptable symptoms ofsevere respiratory disease in the immunized host. The attenuated virusor subviral particle may be present in a cell culture supernatant,isolated from the culture, or partially or completely purified. Thevirus may also be lyophilized, and can be combined with a variety ofother components for storage or delivery to a host, as desired.

The invention further provides novel vaccines comprising aphysiologically acceptable carrier and/or adjuvant and an isolatedattenuated gene position-shifted RSV. In one embodiment, the vaccine iscomprised of a gene position-shifted RSV having at least one, andpreferably two or more attenuating mutations or other nucleotidemodifications as described above. The vaccine can be formulated in adose of 10³ to 10⁶ PFU of attenuated virus. The vaccine may compriseattenuated gene position-shifted RSV that elicits an immune responseagainst a single RSV strain or antigenic subgroup, e.g. A or B, oragainst multiple RSV strains or subgroups. In this regard, geneposition-shifted RSV of the invention can individually elicit amonospecific immune response or a polyspecific immune response againstmultiple RSV strains or subgroups. Gene position-shifted RSV can becombined in vaccine formulations with other RSVs having differentimmunogenic characteristics for more effective protection against one ormultiple RSV strains or subgroups.

In related aspects, the invention provides a method for stimulating theimmune system of an individual to elicit an immune response against oneor more strains or subgroups of RSV in a mammalian subject. The methodcomprises administering a formulation of an immunologically sufficientamount of an attenuated, gene position-shifted RSV in a physiologicallyacceptable carrier and/or adjuvant. In one embodiment, the immunogeniccomposition is a vaccine comprised of gene position-shifted RSV havingat least one, and preferably two or more attenuating mutations or othernucleotide modifications specifying a desired phenotype as describedabove. The vaccine can be formulated in a dose of 10³ to 10⁶ PFU ofattenuated virus. The vaccine may comprise attenuated geneposition-shifted RSV virus that elicits an immune response against asingle RSV strain or antigenic subgroup, e.g. A or B, or againstmultiple RSV strains or subgroups. In this context, the geneposition-shifted RSV can elicit a monospecific immune response or apolyspecific immune response against multiple RSV strains or subgroups.Alternatively, gene position-shifted RSV having different immunogeniccharacteristics can be combined in a vaccine mixture or administeredseparately in a coordinated treatment protocol to elicit more effectiveprotection against one RSV strain, or against multiple RSV strains orsubgroups. Preferably the immunogenic composition is administered to theupper respiratory tract, e.g., by spray, droplet or aerosol. Often, thecomposition will be administered to an individual seronegative forantibodies to RSV or possessing transplacentally acquired maternalantibodies to RSV.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Shift of the G gene, or the F gene, or the G and F genestogether, to a promoter-proximal position within the RSV genome. Thediagram at the top illustrates the RSV genome designated Blp/ΔSH, inwhich the SH gene has been deleted as described previously (Whitehead etal., J. Virol. 73:3438-3442, 1999; incorporated herein by reference) anda BlpI restriction enzyme site has been added to the upstream noncodingregion of the promoter-proximal NS1 gene. The gene-end (GE) signal ofthe M gene has an asterisk to indicate that it contains a singlenucleotide change incorporated during deletion of the SH gene that makesit identical to the naturally-occurring SH GE signal, as describedpreviously (Whitehead et al., J. Virol. 73:3438-3442, 1999; incorporatedherein by reference). The two boxes underneath the diagram show detailsof the structure of genome Blp/ΔSH as well as modifications that weremade to the promoter-proximal end (left hand box) and M-G-F-M2 region(right hand box) of the ΔSH/Blp genome to create genomes G1/ΔSH, F1/ΔSHand G1F2/ΔSH. All manipulations were performed with a cloned cDNA of RSVantigenomic RNA (Collins et al., Proc. Natl. Acad. Sci. USA92:11563-11567, 1995; Collins et al., Virology 259:251-255, 1999; andWhitehead et al., J. Virol. 73:3438-3442, 1999; incorporated herein byreference), and nucleotide positions are numbered according to thecomplete antigenomic sequence of wild type recombinant RSV (containingthe SH gene) (Collins et al., Proc. Natl. Acad. Sci. USA 92:11563-11567,1995; Murphy et al., U.S. Pat. No. 5,993,824; each incorporated hereinby reference). Note that “upstream” and “downstream” refer to thepromoter-proximal and promoter-distal directions, respectively (thepromoter is at the 3′ leader end of negative-sense genomic RNA, which isat the left hand end as drawn in FIG. 1).

Genome Blp/ΔSH: nucleotides 92 and 97 of the previously-describedSH-deletion mutant of RSV (Whitehead et al., J. Virol. 73:3438-3442,1999; incorporated herein by reference) were changed from G and A(indicated in the top diagram in the left hand box with small caseletters), respectively, to C and C (bold capital letters), therebycreating a BlpI site (underlined) one nucleotide in front of the ATGtranslational start codon (italicized, bold) of the NS1 open readingframe (ORF). The M-G-F region of the Blp/ΔSH genome (right hand box)illustrates the SH deletion (the SH gene normally lies between the M andG genes).

Genome G1/ΔSH: This genome contains the G gene in the promoter-proximalposition, inserted into the BlpI site (left hand box). The G cDNA insertwas constructed as follows: the complete G ORF and downstream Gnoncoding sequence and GE signal (nucleotides 4692 to 5596) wereengineered to be followed by a 6-nucleotide IG sequence (representingthe first 6 nucleotides of the naturally-occurring G-F IG sequence,CATATT (SEQ ID NO: 1) followed by a copy of the 10-nucleotide GS signalof the NS1 gene (boxed). This cloned sequence was flanked by BlpI sites,and was cloned into the BlpI site of genome Blp/ΔSH. This placed the GORF, under the control of RSV GS and GE signals, into thepromoter-proximal position. In the same genome, the G gene was deletedfrom its downstream position between the M and F genes (the point ofdeletion is indicated with a large arrow), and the M and F genes werenow separated only by the G-F IG sequence.

Genome F1/ΔSH: This genome contains the F gene in the promoter-proximalposition, inserted into the BlpI site. The F cDNA insert was constructedas follows: the complete F ORF and downstream noncoding sequence and GEsignal (nucleotides 5662 to 7551) were engineered to be followed by a6-nucleotide IG sequence (representing the first 6 nucleotides of thenaturally-occurring F-M2 IG sequence, CACAAT (SEQ ID NO: 2) followed bythe 10-nucleotide GS signal of the NS1 gene. This cloned sequence wasflanked by BlpI sites and was cloned into the BlpI site of genomeBlp/ΔSH. This placed the F ORF, under the control of RSV GS and GEsignals, into the promoter-proximal position. In the same genome, the Fgene was deleted from its downstream position between the G and M2 genes(the point of deletion is indicated with a large arrow), and these twogenes were now separated only by the F-M2 IG sequence.

Genome G1F2/ΔSH: This genome contains the G and F genes in the first andsecond promoter-proximal locations, respectively, inserted as a singlecDNA into the BlpI site. This G-F cDNA insert was constructed to contain(in upstream to downstream order): the complete G ORF, its downstreamnoncoding and GE signal, the G-F IG sequence, the complete F gene, 6nucleotides from the F-M2 IG sequence (CACAAT) (SEQ ID NO: 2), and theNS1 GS signal. This cDNA was flanked by BlpI sites and was cloned intothe BlpI site of genome Blp/ΔSH. This placed the G and F genes intopositions 1 and 2, respectively, relative to the promoter. In the samegenome, the G and F genes were deleted from their downstream positionsbetween the M and M2 genes (the point of deletion is indicated with alarge arrow), and M and M2 genes were now separated only by the F-M2 IGsequence.

FIG. 2. Production of infectious recombinant virus during thetransfection and initial passages in vitro. HEp-2 cells were transfectedwith the indicated individual antigenomic plasmid and the N, P, L andM2-1 support plasmids as described (Murphy et al., U.S. Pat. No.5,993,824). The medium supernatants were harvested 3 days later andsubjected to serial undiluted passage in HEp-2 cells, with the mediumsupernatant harvested at 3- to 7-day intervals. Samples of each harvestwere taken, flash-frozen, and analyzed later in parallel by plaqueassay. The viruses were given the same designations as their respectivecDNAs, i.e. Blp/ΔSH, G1/ΔSH etc. Data are shown for Blp/ΔSH, G1/ΔSH,F1/ΔSH and G1F2/ΔSH. Transfection was at 32° C. and subsequent passageswere at 37° C.

FIG. 3A Replication in vitro of recombinant wt RSV (containing the SHgene), and the ΔSH (not containing a BlpI site), Blp/ΔSH, G1/ΔSH andG1F2/ΔSH mutant viruses following infection at an input multiplicity ofinfection (MOI) of 0.1. Replicate cultures of Vero (top panel) or HEp-2cells (lower panel) were infected and incubated at 37° C. At theindicated time points, duplicate monolayers were harvested for eachvirus and the medium supernatants were flash-frozen. These were analyzedlater in parallel by plaque assay.

FIG. 3B depicts single-step growth of the Blp/SH, G1/SH, F1/SH andG1F2/SH viruses following infection of Hep-2 (top) and Vero (bottom)cell monolayers at an input multiplicity of infection of 3.0. Replicatecell monolayers were infected and incubated at 37° C., and at theindicated time points duplicate monolayers were harvested and the mediumsupernatants were flash-frozen.

FIG. 4. Western blot analysis of the expression of the G protein byBlp/ΔSH, G1/ΔSH and G1F2/ΔSH viruses in Vero cells. The cells from eachtime point in the top panel of FIG. 3A were harvested and the totalprotein was analyzed by gel electrophoresis and Western blotting. Theblots were developed by incubation with a rabbit antiserum specific topeptide of the G protein. Bound antibodies were then visualized bychemiluminescence. The G protein migrates as two forms: the 90 kDamature form and a 50 kDa incompletely-glycosylated form.

FIG. 5. Structures of attenuated RSV's in which the G and F genes havebeen shifted to positions 1 and 2. Panel A: Structure of recombinant RSVG1F2/ΔNS2ΔSH, in which the SH and NS2 genes were deleted as described(Teng and Collins, J. Virol. 73:466-473, 1999; Whitehead et al., J.Virol. 73:3438-3442, 1999; incorporated herein by reference), and the Gand F genes were moved into positions 1 and 2, respectively, asdescribed above in FIG. 1. Panel B: Structure of recombinant RSVG1F2/ΔNS1ΔNS2ΔSH, in which the SH, NS1 and NS2 genes were deleted andthe G and F genes were moved into positions 1 and 2, respectively.

FIG. 6 details construction of a chimeric rBRSV/HRSV genome in which theBRSV G and F genes have been deleted and the G and F genes of HRSV havebeen placed in a promoter-proximal position. BRSV genes are shaded; HRSVgenes are clear. Nucleotide sequence position numbers are relative tothe complete rBRSV antigenome (Buchholz et al., J. Virol. 73:251-259,1999; Buchholz et al., J. Virol. 74:1187-1199, 2000; GenBank accessionnumber AF092942 or complete rHRSV antigenome in Collins et al., Proc.Natl. Acad. Sci. USA 92:11563-11567, 1995; each incorporated herein byreference); sequence position numbers that refer to the HRSV sequenceare underlined. FIG. 6, panel A details structure of rBRSV containingNotI, SalI and XhoI sites that were added in previous work (Buchholz etal., J. Virol. 73:251-259, 1999; Buchholz et al., J. Virol.74:1187-1199, 2000). FIG. 6, panel B depicts modifications to rBRSV tocreate rBRSV/A2-G1F2. The BRSV G and F genes were deleted by digestionwith SalI and XhoI and religation of the resulting compatible cohesiveends. The region of the genome of HRSV from nucleotides 4692 to 7557,containing the G and F genes, was amplified by PCR using primers thatcontained desired changes to be incorporated into each end of the cDNA.The amplified PCR product contained (in upstream to downstream order): aNotI site, a BlpI site, the complete HRSV G ORF, its downstreamnoncoding and GE signal, the HRSV G-F IG sequence, the complete HRSV Fgene, 6 nucleotides from the HRSV F-M2 IG sequence (CACAAT), the NS1 GSsignal, a BlpI site and a NotI site. This cDNA was cloned into theunique Notl site at position 67 of rBRSV. FIG. 6, panel C illustratesstructure of the genomic RNA of rBRSV/A2-G1 F2.

FIG. 7 depicts multicycle growth of RBRSV, rHRSV(rA2), rBRSV/A2, andrBRSV/A2-G1 F2 in HEp-2 human (left panel) and MDBK bovine (right panel)cells. Duplicate cell monolayers were infected with the indicated virusat an MOI of 0.1 and incubated at 37° C., and medium aliquots wereharvested at the indicated times, flash frozen, stored at −70° C. andtitrated later in duplicate. Each value is the mean titer of two wells.

FIG. 8 shows indirect immunofluorescence of HEp-2 cells infected withrBRSV/A2-G1F2, rBRSV/A2, or rA2. Cells were infected at an MOI of 0.1,incubated at 37° C. for 96 hours, fixed with acetone, permeabilized andreacted with monoclonal antibody 021/1G, specific to the G protein ofHRSV, or with monoclonal antibody 44F, specific to the F protein ofHRSV. Antibody binding was visualized by reaction with a tagged antibodyspecific to murine IgG.

FIG. 9 details construction of a chimeric rBRSV/HRSV containing the M, Gand F genes of HRSV. BRSV genes are shaded; HRSV genes are clear.Sequence position numbers that refer to HRSV genes are underlined. FIG.9, panel A depicts modification of rBRSV/A2 to contain a unique MluIsite at position 3204, within the intergenic region between the P and Mgenes (P-M IG). The sequence of the IG is shown, with small case lettersindicating the original nucleotide assignments. The underlined lettersindicate the Mlul site created by the 5 nucleotide substitutions.Nucleotide sequence position numbers are relative to the complete rBRSVantigenome (Buchholz et al., J. Virol. 73:251-259, 1999; Buchholz etal., J. Virol. 74:1187-1199, 2000; GenBank accession number AF092942 orcomplete rHRSV antigenome in Collins et al., Proc. Natl. Acad. Sci. USA92:11563-11567, 1995); sequence position numbers that refer to the HRSVsequence are underlined. FIG. 9, panel B illustrates modification ofrBRSV/A2 to create rBRSV/A2-MGF. The Mlul-SalI fragment containing theBRSV M and SH genes was excised and replaced with an MluI-SalI fragmentcontaining the HRSV M gene. The MluI-SalI fragment containing the HRSV Mgene is shown in the box. Also shown is the sequence immediatelyupstream of the MluI site, including the BRSV P gene-end sequence, andthe sequence immediately downstream of the SalI site, including theintergenic sequence between the M and G genes (M-G IG), the HRSV Ggene-start signal, and the ATG (bold, italics) that begins the G ORF.FIG, 9, panel C depicts the structure of the genome of rBRSV/A2-MGF.

FIG. 10 depicts the structures of the genomes of recombinant BRSV(rBRSV, top) and five BRSV/HRSV chimeric viruses in which specific BRSVgenes (shaded rectangles) were replaced with their HRSV counterparts(open rectangles). In addition, in the bottom two viruses the G and Fgenes were moved from their normal positions to positions 3 and 4 or 1and 2. In the diagram of rBRSV, several restriction sites are indicated.Restriction sites used in the various constructions are indicated: theKpnl site occurs naturally and the others were introduced as necessary(Buchholz, et al., J. Virol. 73:251-9, 1999; Buchholz, et al. J. Virol.74:1187-1199, 2000, each incorporated herein by reference).

FIG. 11 depicts multicycle growth of the BRSV/HRSV chimeric virusesrBRSV/A2-G3F4 (top panel) and HEx (bottom panel) compared to rHRSV (rA2)and rBRSV parental viruses as well as the previously-described chimericviruses rBRSV/A2-GF (previously called rBRSV/A2; Buchholz, 2000, supra)and rBRSV/A2-G1F2. Monolayer cultures of Vero cells were infected at aninput multiplicity of infection of 0.1 and incubated at 37° C. Samplesfrom the overlying medium were harvested at the indicated times andvirus titers were determined by the limiting dilution method. To 0.1 mlof serial 10-fold virus dilutions per well, 104 BHK-21 cells were addedin a 0.1 ml volume. After 48 hours, cells were fixed in 80% acetone, andan indirect immunofluorescence assay using an antibody specific to theBRSV M protein, cross-reactive with the HRSV M protein, was done, andfoci of infected cells were counted (see, Buchholz et al., J. Virol.73:251-9, 1999 (incorporated herein by reference).

FIG. 12 compares the size of the plaque or focus formed by the indicatedviruses on human HEp-2 cells (top panel) versus bovine MDBK cells(bottom panel), expressed as a percentage compared to HRSV (HEp-2 cells)or BRSV (MDBK cells).

FIGS. 13A and 13B depict the structures of the genomes of recombinantBRSV in which the N and/or P genes were replaced by their HRSVcounterparts. FIG. 13A shows chimeras in which these substitutions weremade in the rBRSV backbone, and FIG. 13B shows chimeras in which thebackbone was the rBRSV/A2-NS1+2 virus.

FIG. 14 Diagram of the genomic RNA of the recombinant rRSV/6120 viruscontaining a deletion in the SH gene, drawn as the negative sense RNA,3′ to 5′, with each encoded mRNA indicated with a rectangle and nonmRNA-coding extragenic and intergenic regions as a horizontal line. Theparent RSV antigenomic cDNA was as described previously (Collins, et al,Proc. Natl. Acad. Sci. USA 92:11563-11567, 1995), with the furthermodification of an XmaI site in the G-F intergenic region (Bukreyev, etal., J. Virol. 70:6634-6641, 1996, incorporated herein by reference).This cDNA was modified (i) to contain five translationally-silentnucleotide substitutions in the last four codons of the SH ORF includingthe translational stop codon and (ii) to delete 112 nucleotides(positions 4499-4610) of the complete antigenomic sequence from thedownstream non-translated region of the SH gene (box). The XhoI and PacIsites used in the construction are italicized and labeled, the SHgene-end signal is underlined, the SH codons are shown as triplets,nucleotide substitutions are in small case, and the deleted sequence isrepresented with a box with the sequence positions indicated.

FIGS. 15A-15B Growth kinetics of rRSV/6120, containing a deletion in theSH gene, compared to its full length recombinant rRSV parent D53. Threesets (FIG. 15A, FIG. 15B and FIG. 15C) of monolayer cultures of HEp-2cells were infected with the indicated virus at an input multiplicity ofinfection of 0.005. Following an adsorption period, cells were incubatedat 37° C. At 12 h intervals, the medium was harvested in its entiretyand aliquots were flash-frozen for later titration. The cells werewashed three times and fresh medium was added and the incubationcontinued. At the end of the experiment, the samples were analyzed byplaque assay to determine virus titer.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides recombinant respiratory syncytial viruses(RSVs) which are modified by shifting a gene order or spatial positionof one or more genes within a recombinant RSV genome or antigenome togenerate a vaccine virus that is infectious and attenuated in humans andother mammals. Typically, the recombinant RSV genome or antigenome ismodified by repositioning one or more “shifted” genes or gene segments,directly or indirectly via introduction, deletion or rearrangement of asecond, “displacement polynucleotide” within the genome, resulting in apositional shift of the subject “shifted” gene or genome segment to amore promoter-proximal or promoter-distal position. Shifting of gene orgenome segment positions in this context is determined to a position ofthe subject gene or genome segment in a parent RSV genome or antigenomeprior to introduction of the gene shift, for example relative to theposition of the subject gene or genome segment in a wild type RSV genomeor antigenome (e.g., HRSV A2 or BRSV kansas strains) or in a parentalrecombinant RSV genome or antigenome as disclosed herein, prior to thegene shift.

In certain aspects of the invention, the gene position-shifted RSVfeatures one or more shifted genes or genome segments that are shiftedto a more promoter-proximal or promoter-distal position by insertion,deletion, or rearrangement of one or more displacement polynucleotideswithin the partial or complete recombinant RSV genome or antigenome. Thedisplacement polynucleotides may comprise an RSV gene or genome segment,including an RSV gene or genome segment from a different or“heterologous” RSV (e.g., in the case of a heterologous gene or genomesegment inserted into genome or antigenome of a different RSV).Alternatively, the displacement polynucleotides may be from a non-RSVsource, including from a non-RSV pathogen such as parainfluenza virus(PIV) or measles virus. The displacement polynucleotides may encode aprotein or a portion of a protein, such an immunogenic domain or epitopeof a glycoprotein, or they may represent an incomplete or impairedcoding sequence, including non-coding and nonsense polynucleotidesequences.

In certain aspects of the invention, the recombinant RSV features one oror positionally shifted genes or genome segments that may be shifted toa more promoter-proximal or promoter-distal position by insertion,deletion, or rearrangement of one or more displacement polynucleotideswithin the partial or complete recombinant RSV genome or antigenome. Incertain aspects, the displacement polynucleotides are RSV genes orgenome segments. In other aspects, displacement polynucleotides lack acomplete open reading frame (ORF). Within more detailed embodiments,displacement polynucleotides comprise polynucleotide inserts of between150 nucleotides (nts) and 4,000 nucleotides in length. Displacementpolynucleotides may be inserted or rearranged into a non-coding region(NCR) of the recombinant genome or antigenome, or may be incorporated inthe recombinant RSV genome or antigenome as a separate gene unit (GU).

Gene position shifts within the recombinant RSV of the invention aretypically determined relative to the genome or antigenome “promoter”.The RSV promotor contains the polymerase initiation site, which is aconserved sequence element recognized by the polymerase. The promoter islocated at the 3′ end of the genome or antigenome, within approximatelythe thirty 3′-terminal nucleotides. In the case of the RSV genome, thepromoter directs both transcription and replication. However, theantigenome “promoter” which lacks transcription signals and onlynaturally controls replication can be modified to direct transcriptionby insertion of known transcription signals. For the purposes ofdescribing the invention, the RSV promotor is thus construed to resideat the 3′ end of either the genome or antigenome, whereby the terms“promoter-proximal” and “promoter-distal” used herein alternately referto a direction toward, or away from, respectively, the 3′ end of thegenome or antigenome.

Thus provided within the invention are isolated polynucleotidemolecules, vectors (expression constructs), and recombinant virusesincorporating a recombinant RSV genome or antigenome—wherein one or moregenes or gene segments is/are shifted to a more promoter-proximal orpromoter-distal position within the recombinant genome or antigenomecompared to a parental or wild type position of the gene in the RSV genemap. Shifting the position of genes in this manner provides for aselected increase or decrease in expression of one or more positionally“shifted” genes, depending on the nature and degree of the positionalshift. In one embodiment, RSV glycoproteins are upregulated by shiftingone or more glycoprotein-encoding genes to a more promoter-proximalposition. Genes of interest for manipulation to create geneposition-shifted RSV include any of the NS1, NS2, N, P, M, SH, M2(ORF1),M2(ORF2), L, F or G genes or a genome segment that may be part of a geneor extragenic. A variety of additional mutations and nucleotidemodifications are provided within the gene position-shifted RSV of theinvention to yield desired phenotypic and structural effects.

The recombinant construction of human-bovine RSV yields a viral particleor subviral particle that is infectious in mammals, particularly humans,and useful for generating immunogenic compositions for clinical use.Also provided within the invention are novel methods and compositionsfor designing and producing attenuated, gene position-shifted RSV, aswell as methods and compositions for the prophylaxis and treatment ofRSV infection. Gene position-shifted RSV and immunogenic compositionsaccording to the invention may elicit an immune response to a specificRSV subgroup or strain, or they may elicit a polyspecific responseagainst multiple RSV subgroups or strains. Gene position-shifted RSV ofthe invention are thus infectious and attenuated in humans and othermammals. In related aspects, the invention provides novel methods fordesigning and producing attenuated, gene position-shifted RSV that areuseful in various compositions to generate a desired immune responseagainst RSV in a host susceptible to RSV infection. Included withinthese aspects of the invention are novel, isolated polynucleotidemolecules, vectors, and infected cells incorporating such molecules thatcomprise a gene position-shifted RSV genome or antigenome. Geneposition-shifted RSV according to the invention may elicit an immuneresponse to a specific RSV subgroup or strain, or a polyspecificresponse against multiple RSV subgroups or strains. Yet additionalcompositions and methods are provided for designing and producingattenuated, gene position-shifted RSV as vectors for incorporatingantigenic determinants of other pathogens to generate a desired immuneresponse against different pathogens of interest. Also provided withinthe invention are methods and compositions incorporating geneposition-shifted RSV for prophylaxis and treatment of infection anddisease caused by RSV and other pathogens.

The present invention culminates and supplements a continuing line ofdiscovery founded upon the recent advent and refinement of methods forproducing infectious recombinant RSV from cDNA. Based upon this work, ithas been possible to directly investigate the roles of RNA and proteinstructures in RSV gene expression and replication. These investigationsare described or reported in U.S. Provisional Patent Application No.60/007,083, filed Sep. 27, 1995; U.S. patent application Ser. No.08/720,132, filed Sep. 27, 1996; U.S. Provisional Patent Application No.60/021,773, filed Jul. 15, 1996; U.S. Provisional Patent Application No.60/046,141, filed May 9, 1997; U.S. Provisional Patent Application No.60/047,634, filed May 23, 1997; U.S. Pat. No. 5,993,824, issued Nov. 30,1999 (corresponding to International Publication No. WO 98/02530); U.S.patent application Ser. No. 09/291,894, filed by Collins et al. on Apr.13, 1999; U.S. Provisional Patent Application No. 60/129,006, filed byMurphy et al. on Apr. 13, 1999; Crowe et al., Vaccine 12: 691-699, 1994;and Crowe et al., Vaccine 12: 783-790, 1994; Collins, et al., Proc Nat.Acad. Sci. USA 92:11563-11567, 1995; Bukreyev, et al., J Virol70:6634-41, 1996, Juhasz et al., J. Virol. 71(8):5814-5819, 1997; Durbinet al., Virology 235:323-332, 1997; Karron et al., J. Infect. Dis.176:1428-1436, 1997; He et al. Virology 237:249-260, 1997; Baron et al.J. Virol. 71:1265-1271, 1997; whitehead et al., Virology 247(2):232-9,1998a; Whitehead et al., J. Virol. 72(5):4467-4471, 1998b; Jin et al.Virology 251:206-214, 1998; Bukreyev, et al., Proc. Nat. Acad. Sci. USA96:2367-2372, 1999; Bermingham and Collins, Proc. Natl. Acad. Sci. USA96:11259-11264, 1999 Juhasz et al., Vaccine 17:1416-1424, 1999; Juhaszet al., J. Virol. 73:5176-5180, 1999; Teng and Collins, J. Virol.73:466-473, 1999; Whitehead et al., J. Virol. 73:9773-9780, 1999;Whitehead et al., J. Virol. 73:871-877, 1999; and Whitehead et al., J.Virol. 73:3438-3442, 1999, Jin, et al., Virology 273:210-8, 2000; Jin,et al., J. Virol. 74:74-82, 2000; Teng, et al., J. Virol. 74:9317-21,2000, each of which are incorporated herein by reference in theirentirety for all purposes).

With regard to gene position-shifted RSV of the invention, a number ofthe foregoing incorporated disclosures have focused on modification ofthe naturally-occurring gene order in RSV. For example, each of the NS1,NS2, SH and G genes have been successfully deleted individually ininfectious RSV recombinants, thereby shifting the position of downstreamgenes relative to the viral promoter. In other recombinants within theinvention, the NS1 and NS2 gene were deleted together, shifting theremaining genes in promoter-proximal direction within the recombinantRSV genome or antigenome. For example, when NS1 and NS2 are deletedtogether, N is moved from position 3 to position 1, P from position 4 toposition 2, and so on. Deletion of any other RSV gene within alternateembodiments of the invention will similarly shift the position (relativeto the promoter) of those genes which are located further downstream.For example, SH occupies position 6 in wild type virus, and its deletiondoes not affect M at position 5 (or any other upstream gene) but moves Gfrom position 7 to 6 relative to the promoter. It should be noted thatgene deletion also can occur (rarely) in a biologically-derived mutantvirus (Karron et al., Proc. Natl. Acad. Sci. USA 94:13961-13966, 1997;incorporated herein by reference). Note that “upstream” and “downstream”refer to the promoter-proximal and promoter-distal directions,respectively (the promoter is at the 3′ leader end of negative-sensegenomic RNA).

Gene order shifting modifications (i.e., positional modifications movingone or more genes to a more promoter-proximal or promoter-distallocation in the recombinant viral genome) with gene position-shifted RSVof the invention result in viruses with altered biological properties.For example, RSV lacking NS1, NS2, SH, G, NS1 and NS2 together, or SHand G together, have been shown to be attenuated in vitro, in vivo, orboth. It is likely that this phenotype was due primarily to the loss ofexpression of the specific viral protein. However, the altered gene mapalso likely contributed to the observed phenotype. This effect iswell-illustrated by the SH-deletion virus, which grew more efficientlythan wild type in some cell types, probably due to an increase in theefficiency of transcription, replication or both resulting from the genedeletion and resulting change in gene order and possibly genome size. Inother viruses, such as RSV in which NS1 and/or NS2 were deleted, alteredgrowth that might have occurred due to the change in gene order likelywas obscured by the more dominant phenotype due to the loss ofexpression of the RSV protein(s).

Yet additional changes have been successfully introduced to change thegene order of RSV to improve its properties as a live-attenuatedvaccine. In specific examples demonstrating efficacy of the invention,the RSV G and F genes were shifted, singly and in tandem, to a morepromoter-proximal position relative to their wild-type gene order. Thesetwo proteins normally occupy positions 7 (G) and 8 (F) in the RSV geneorder (NS1-NS2-N-P-M-SH-G-F-M2-L). In order to increase the possibilityof successful recovery, these positional manipulations of G and F wereperformed in a version of RSV in which the SH gene had been deleted(see, e.g., Whitehead et al., J. Virol., 73:3438-42 (1999), incorporatedherein by reference). This facilitates viral recovery because this virusmakes larger plaques in vitro (Bukreyev et al., J. Virol. 71:8973-82,1997, incorporated herein by reference). G and F were then movedindividually to position 1, or were moved together to positions 1 and 2,respectively.

Surprisingly, recombinant RSV were readily recovered in which G or Fwere moved to position 1, or in which G and F were moved to positions 1and 2, respectively. This result differed greatly from previous reportedstudies with vesicular stomatitis virus (VSV), where movement of thesingle VSV glycoprotein gene by only two positions was very deleteriousto virus growth (Ball et al., J. Virol. 73:4705-4712, 1999, incorporatedherein by reference). The ability to recover these altered viruses alsowas surprising because RSV replicates inefficiently and because RSV hasa complex gene order and movement of the glycoprotein genes involved alarge number of position changes. Indeed, the rearranged RSV's grew atleast as well as their immediate parent having the wild type order ofgenes. As indicated above, this is particularly important for RSV, sincethe wild type virus grows inefficiently in cell culture and a furtherreduction in replication in vitro would likely render vaccinepreparation unfeasible. Thus, it is remarkable that all of theNS1-NS2-N-P-M proteins could be displaced by one or two positionsrelative to the promoter without a significant decrease in growthfitness. In addition, examination of the expression of the Gglycoprotein showed that it was increased up to several-fold over thatof its parent virus. This indicated that a vaccine virus containing Gand/or F in the first position expresses a higher molar amount of theseprotective antigens compared to the other viral proteins, and thusrepresent a virus with desired vaccine properties.

Similarly extensive modifications in gene order also were achieved withtwo highly attenuated RSV vaccine candidates in which the NS2 gene wasdeleted on its own, or in which the NS1 and NS2 genes were deletedtogether, as described in more detail in the above-incorporatedreferences. In these two vaccine candidates, the G and F glycoproteinswere moved together to positions 1 and 2 respectively, and the G, F andSH glycoproteins were deleted from their original downstream position.Thus, the recovered viruses G1F2ΔNS2ΔSH and G1F2/ΔNS1ΔNS2ΔSH had two andthree genes deleted respectively in addition to the shift of the G and Fgenes. To illustrate the extent of the changes involved, the gene ordersof wild type RSV (NS1-NS2-N-P-M-SH-G-F-M2-L) and the G1F2/ΔNS2ΔSH virus(G-F-NS1-N-P-M-M2-L) or the ΔNS1ΔNS2ΔSH (G-F-N-P-M-M2-L) can becompared. This shows that the positions of most or all of the genesrelative to the promoter were changed. Nonetheless, these highlyattenuated derivatives retained the capacity to be grown in cellculture.

Yet additional changes have been successfully introduced to change thegene order of RSV in a human-bovine chimeric RSV to improve itsproperties as a live-attenuated vaccine (See, U.S. patent applicationSer. No. 09/602,212, filed by Bucholz et al. on Jun. 23, 2000, itscorresponding PCT application published as WO 01/04335 on Jan. 18, 2001,and its priority provisional U.S. Application No. 60/143,132 filed onJul. 9, 1999, each incorporated herein by reference). As illustrated inthe examples below, an infectious recombinant human-bovine chimeric RSV(rBRSV/HRSV) was successfully constructed and recovered in which theHRSV G and F genes are substituted into a recombinant bovine RSV (rBRSV)background. The resulting human-bovine chimera contains two genes ofHRSV, namely G and F, and eight genes from BRSV, namely NS1, NS2, N, P,M, SH, M2 and L. In addition to this basic substituted glycoproteinconstruction, the HRSV G and F genes are shifted to a morepromoter-proximal position in the rBRSV backbone, i.e., relative to thewild-type gene order position of the F and G genes in the RSV genome.More specifically, the F and G genes were moved from their usuallocation relative to the promoter, namely gene positions 7 and 8,respectively, to positions 1 and 2, respectively. The resulting chimericrecombinant virus, rBRSV/A2-G1F2, is very similar in its levels of F andG protein expression as detected by immunofluorescence to that of wtHRSV, which result is interpreted to show increased expression of the Gand F glycoproteins attributed to the promoter-proximal shift of thegenes. Since the present rBRSV/A2-G1F2 virus bears the sameconstellation of BRSV genes in its genetic background, it is likely toshare this strong host range restriction phenotype. In this context, theincreased expression of the two protective antigens in vivo willincrease the immunogenicity of this virus to produce highly desirablevaccine properties.

RSV is a nonsegmented negative strand RNA virus of OrderMononegavirales. The mononegaviruses constitute a large and diverseOrder that includes four families: Family Rhabdoviridae, represented byvesicular stomatitis virus (VSV) and rabies virus; Family Bornaviridae,represented by borna disease virus; Family Filoviridae, represented byMarburg and Ebola viruses, and Family Paramyxoviridae. This latterfamily is further divided into two subfamilies: Paramyxovirinae, whichincludes Sendai, measles, mumps and parainfluenza viruses, andPneumovirinae, which includes respiratory syncytial virus.

The genome of a mononegavirus is a single strand of RNA that containsfrom 5 (VSV) to 11 (RSV) genes arranged in a linear array. Themononegavirus genome does not encode protein directly (hence thedesignation “negative sense”), but rather encodes complementarypositive-sense mRNAs that each encode one or more proteins. Typically, agene begins with a short gene-start signal and ends with a shortgene-end signal. These signals usually consist of 8 to 12 nucleotidesand usually are highly conserved between genes of a given virus and to alesser extent between related viruses.

In the case of RSV, the genome is more than 15.2 kb in length and istranscribed into 10 separate major mRNAs that encode 11 identifiedproteins. Specifically, the RSV gene order is3′-NS1-NS2-N-P-M-SH-G-F-M2-L-5′, and the M2 mRNA encodes two proteins,M2-1 and M2-2 from overlapping ORFs. The gene-start and gene-end signalsof RSV, together with sequences involved in RNA replication and inpromoter function, also have been identified and analyzed in ongoingwork (Bukreyev et al., J. Virol. 70:6634-6641, 1996; Collins et al.,Proc. Natl. Acad. Sci. USA 88:9663-9667, 1991; Mink et al., Virology185:615-624, 1991; Grosfeld et al., J. Virol. 69:5677-5686, 1995; Hardyand Wertz, J. Virol. 72:520-526, 1998; Hardy et al., J. Virol.73:170-176, 1999; Kuo et al., J. Virol. 70:6143-6150, 1996; Kuo et al.,J. Virol. 70:6892-6901, 1996; Samal and Collins, J. Virol. 70:5075-5082,1996; Kuo et al., J. Virol. 71:4944-4953, 1997; Fearns and Collins, J.Virol. 73:388-397, 1999; each incorporated herein by reference).

The 3′ end of a mononegavirus genome contains a promoter that directsentry of the polymerase Lamb and Kolakofsky, Fields Virology1:1177-1204, 1996; and Wagner and Rose, Fields Virology 121-1136, 1996;each incorporated herein by reference). This promoter is containedcompletely or in part in an extragenic leader region at the 3′ end ofthe genome. The polymerase then transcribes the genome 3′-to-5′ in alinear, stop-restart manner guided by the gene-start and gene-endsignals. The gene-start signal of each gene directs initiation of thesynthesis of the corresponding mRNA and the gene-end signal directspolyadenylation, termination and release of the corresponding mRNA. Thepolymerase then remains template-bound and reinitiates at the nextdownstream gene-start signal. This process is repeated to transcribe onegene after another in their 3′-to-5′ order (Lamb and Kolakofsky, FieldsVirology 1:1177-1204, 1996; Wagner and Rose, Fields Virology, 1121-1136,1996; Abraham and Banerjee, Proc. Natl. Acad. Sci. USA 73:1504-1508,1976; Ball and White, Proc. Natl. Acad. Sci. USA, 73:442-446, 1976;Ball, J. Virol. 21:411-414, 1977; Banerjee et al., J. Gen. Virol.34:1-8, 1977; Iverson and Rose, Cell 23:477-484, 1981; Iverson and Rose,J. Virol. 44:356-365, 1982; Banerjee et al., Pharmacol. Ther. 51:47-70,1991; each incorporated herein by reference).

Four of the RSV proteins enumerated above are nucleocapsid/polymeraseproteins, namely the major nucleocapsid N protein, the phosphoprotein P,and polymerase protein L, and the transcription antitermination proteinM2-1. Three are surface glycoproteins, namely the attachment G protein,the fusion F glycoprotein responsible for penetration and syncytiumformation, and the small hydrophobic SH protein of unknown function. Thematrix M protein is an internal virion protein involved in virionformation. There are two nonstructural proteins NS1 and NS2 of unknownfunction. Finally, there is a second open reading frame (ORF) in the M2mRNA which encodes an RNA regulatory factor M2-2.

The G and F proteins are the major neutralization and protectiveantigens (Collins, et al., Fields Virology 2:1313-1352, 1996; Connors,et al., J. Virol. 66:1277-81, 1992). Resistance to reinfection by RSV islargely mediated by serum and mucosal antibodies specific against theseproteins. RSV-specific cytotoxic T cells are also induced by RSVinfection and can be directed against a number of different proteins,but this effector has not yet been shown to be an important contributorto long term resistance to reinfection. However, both CD8+ and CD4+cells can be important in regulating the immune response, and both maybe involved in viral pathogenesis (Johnson, et al., J. Virol.72:2871-80, 1998; Srikiatkhachorn and Braciale, J. Exp. Med. 186:421-32,1997). Thus, F and G are the most important antigenic determinants, butother proteins can also play important roles in the immune response.

RSV isolates can be segregated into two antigenic subgroups, A and B, byreactivity with monoclonal antibodies (Anderson, et al., J. Infect. Dis.151:626-33, 1985, Mufson, et al., J. Gen. Virol. 66:2111-24, 1985). Thetwo subgroups exhibit differences across the genome, but are the mostdivergent in the ectodomain of the G protein where the percent aminoacid sequence divergence can exceed 50% and the antigenic divergence is95% based on reactivity of monospecific polyclonal antisera (Johnson, etal., Proc. Natl. Acad. Sci. USA 84:5625-9, 1987; Johnson, et al., J.Virol. 61:3163-6, 1987). The F protein is approximately 10% divergent byamino acid sequence and 50% divergent antigenically between RSV A and Bsubgroups (Johnson, et al., J. Virol. 61:3163-6, 1987; Johnson andCollins, J. Gen. Virol. 69:2623-8, 1988). Thus, both subgroups should berepresented in a vaccine.

RSV and other mononegaviruses have been reported to exhibit a gradientof decreasing gene transcription, such that the most promoter-proximalgene is transcribed the most efficiently, and each gene thereafterdisplays an incrementally-decreasing efficiency of transcription (Lamband Kolakofsky, Fields Virology, 1:1177-1204, 1996; Wagner and Rose,Fields Virology 1121-1136, 1996; Abraham and Banerjee, Proc. Natl. Acad.Sci. USA 73:1504-1508, 1976; Ball and White, Proc. Natl. Acad. Sci. USA73:442-446, 1976; Ball, J. Virol. 21:411-414, 1977; Banerjee et al., J.Gen. Virol., 34:1-8, 1977; Iverson and Rose, Cell 23:477-484, 1981;Iverson and Rose, J. Virol. 44:356-365, 1982; Banerjee et al.,Pharmacol. Ther. 51:47-70, 1991; each incorporated herein by reference).This gradient of gene expression has been reported and partiallycharacterized for RSV (Collins and Wertz, Proc. Natl. Acad. Sci. USA80:3208-3212, 1983; Collins et al., J. Virol. 49:572-578, 1984; Dickenset al., J. Virol. 52:364-369, 1984; each incorporated herein byreference). This gradient is thought to be partially attributed to“fall-off” of the polymerase during sequential transcription. Studieswith the rhabdovirus VSV, one of the simplest of the mononegaviruses,suggest that fall-off occurs primarily at the intergenic regions(Iverson and Rose, Cell 23:477-484, 1981; Iverson and Rose, J. Virol.44:356-365, 1982; each incorporated herein by reference), although thedisproportionately low abundance of the large L mRNA suggests that therealso is significant fall-off within genes.

The gradient of gene transcription reported among mononegaviruses isthought to be a major factor that determines the relative molar ratiosof the various viral mRNAs within an infected cell. This phenomenon inturn is thought to be a major factor determining the relative molarratios of viral proteins. All mononegaviruses have genes encoding thefollowing five proteins or counterparts thereof: an RNA-bindingnucleocapsid protein N, a phosphoprotein P, an internal virion matrixprotein M, an attachment protein G, HA, or HN, and a large polymeraseprotein L. Furthermore, these are always found in the 3′-to-5′ orderN-P-M-G-L. One interpretation is that this genomic organization reflectsa common need among the mononegaviruses for large amounts of the N and Pproteins, a small amount of L, and intermediate amounts of M and G.Alternatively, it has been suggested that the lack of homologousrecombination in mononegaviruses has resulted in the retention of anancestral gene order that is not necessarily optimal for the virus (Ballet al., J. Virol., 73:4705-4712, 1999; incorporated herein byreference).

The constrained gene order and polar nature of transcription has beenproposed as an important factor in the regulation of gene expressionamong mononegaviruses. However, other secondary factors are alsobelieved to affect the relative levels of expression of one or more ofthe mononegaviral proteins, including differences in the efficiencies ofcis-acting RNA signals, differences in efficiencies of translation ofvarious mRNAs, and differences in processing and stability of proteins.

The simple, prototypic mononegavirus, VSV, has 5 genes (Wagner and Rose,Fields Virology 1121-1136, 1996; Schubert et al., J. Virol. 51:505-514,1984; each incorporated herein by reference). However, othermononegaviruses have as many as 11 (RSV) or 12 (pneumonia virus of mice)genes (Barr et al., J. Virol. 68:5330-5334, 1994; incorporated herein byreference). These include proteins such as the fusion F gene found inall paramyxoviruses and pneumoviruses, the C, D and V genes found insome paramyxoviruses, and the NS1, NS2, SH and M2 genes found in mostpneumoviruses. Also, even among the five proteins that may be common toVSV and RSV (N, P, M, G and L), there is clear sequence relatedness onlyfor L, and that relatedness is low (Poch et al., Embo. J. 8:3867-3674,1989; Stec et al., Virology 183:273-287, 1991; each incorporated hereinby reference).

Given this extensive difference in the array and structure of geneproducts, taken together with differences in the structure and functionof trans- and cis-acting components, the features and properties of onemononegavirus, e.g., VSV, cannot be directly extrapolated to othermononegaviruses, such as RSV. This uncertainty is exemplified by thefinding that the RSV polymerase consists of 4 rather than 3 proteins,with the additional one being the M2-1 transcription antiterminationfactor that has no counterpart in VSV (Hardy and Wertz, J. Virol.72:520-526, 1998; Hardy et al., J. Virol. 73:170-176, 1999; Collins etal., Proc. Natl. Acad. Sci. USA 93:81-85, 1996; Fearns and Collins, J.Virol. 73:5852-5864, 1999; each incorporated herein by reference). Thisproved to be critical for the efficient recovery of recombinant RSV(Collins et al., Virology 259:251-255, 1999; incorporated herein byreference). In addition, RSV RNA synthesis is further regulated by atleast two proteins, NS1 and M2-2, which do not have counterparts in VSV(Bermingham and Collins, Proc. Natl. Acad. Sci. USA 96:11259-64, 1999;Whitehead et al., J. Virol. 73:3438-3442, 1999; Atreya et al., J. Virol.72:1452-61, 1998; Jin et al., J. Virol. 74:74-82, 2000; eachincorporated herein by reference).

A number of important features of VSV gene expression and regulationalso do not appear to have any relevance to many other mononegavirusesand in particular to RSV, such as the involvement of terminalcomplementarily in control of VSV gene expression (Wertz et al., Proc.Natl. Acad. Sci. USA 91:8587-8591, 1994; Whelan and Wertz, J. Virol.73:297-306, 1999; Peeples and Collins, J. Virol. 74:146-155, 2000; eachincorporated herein by reference), the highly conserved VSV intergenicregions that are directly involved in gene expression (Barr et al., J.Virol. 71:1794-1801, 1997; incorporated herein by reference) whereasthose of RSV are not (Kuo et al., J. Virol. 70:6143-6150, 1996;incorporated herein by reference), a VSV genomic packaging signal(Whelan and Wertz, J. Virol. 73:307-315, 1999; incorporated herein byreference) not found in other mononegavirus groups, and a control of VSVgene expression and replication by N protein (Wagner and Rose, FieldsVirology, 1121-1136, 1996; Fearns et al., Virology 236:188-201, 1997;incorporated herein by reference). These features do not appear to occurin RSV (Peeples and Collins, J. Virol. 74:146-155, 2000; incorporatedherein by reference), while in contrast RSV has important features ofgene expression and regulation that do not occur in VSV, includingdivergent intergenic sequences that lack cis-acting functional elements(Kuo et al., J. Virol. 70:6143-6150, 1996; incorporated herein byreference), a gene overlap that mediates site-specific attenuation(Fearns and Collins, J. Virol. 73:388-397, 1999; Collins et al., Proc.Natl. Acad. Sci. USA 84:5134-5138, 1987; incorporated herein byreference), and the existence of a polymerase back-tracking mechanismcritical for expression of the L gene (Fearns and Collins, J. Virol.73:388-397, 1999; incorporated herein by reference). In particular, theexistence of a transcription antitermination factor that modulatessequential transcription (Fearns and Collins, J. Virol. 73:5852-5864,1999; incorporated herein by reference), specific regulatory proteins,site-specific attenuation, and gene-junction-specific modulation oftranscription (Hardy et al., J. Virol. 73:170-176, 1999; incorporatedherein by reference) contrast sharply with the situation with VSV.

In certain embodiments of the invention, gene position shifts areachieved by deletion of one or more of the RSV NS1, NS2, SH and/or Ggenes, which deletions are disclosed individually in theabove-incorporated references. Alternative gene or genome segmentdeletions can be constructed involving any of the above identified RSVgenes or genome segments, to alter gene position for remaining RSVgenes. In more detailed embodiments, multiple genes or genome segmentsare deleted, as exemplified in the above-incorporated references bypairwise deletion of the NS1 and NS2 genes (Bukreyev et al., J. Virol.71:8973-8982, 1997; Teng and Collins, J. Virol. 73:466-473, 1999;Whitehead et al., J. Virol. 73:3438-3442, 1999; incorporated herein byreference).

Deletion of one or more genes or genome segments within a recombinantRSV genome or antigenome has the effect of moving all downstream genescloser to the promoter (e.g., by shifting the downstream genes one ormore gene positions in a promoter-proximal direction). For example, theRSV NS1 and NS2 genes are the first and second genes in the genome map,and their coordinate deletion alters the position of all of theremaining genes. Thus, when NS1 and NS2 are deleted together, N is movedfrom position 3 to position 1, P from position 4 to position 2, and soon. Alternatively, deletion of any other gene within the gene order willaffect the position (relative to the promoter) only of those genes whichare located even further downstream. For example, SH occupies position 6in wild type virus, and its deletion does not affect M at position 5 (orany other upstream gene) but moves G from position 7 to 6 relative tothe promoter. It should be noted that gene deletion also can occur(rarely) in biologically-derived virus. For example, a subgroup B RSVthat had been passaged extensively in cell culture spontaneously deletedthe SH and G genes (Karron et al., Proc. Natl. Acad. Sci. USA94:13961-13966, 1997; incorporated herein by reference). Note that“upstream” and “downstream” refer to the promoter-proximal andpromoter-distal directions, respectively (the promoter is at the 3′leader end of negative-sense genomic RNA).

A second example of gene order rearrangement useful within the inventioninvolves the insertion of a gene, genome segment or heterologouspolynucleotide sequence into the recombinant RSV genome or antigenome toalter gene order or introduce a promoter-relative gene position shift inthe recombinant genome or antigenome (see, e.g., Bukreyev et al., J.Virol. 70:6634-6641, 1996; Bukreyev et al., Proc. Natl. Acad. Sci. USA96:2367-2372, 1999; Moriya et al., FEBS Lett. 425:105-111, 1998; Singhand Billeter, J. Gen. Virol. 80:101-106, 1999; incorporated herein byreference). Each inserted gene displaces all downstream genes by oneposition relative to the promoter. These and other displacementpolynucleotides may be inserted or rearranged into a non-coding region(NCR) of the recombinant genome or antigenome, or may be incorporated inthe recombinant RSV genome or antigenome as a separate gene unit (GU).

As used herein, “RSV gene” generally refers to a portion of the RSVgenome encoding an mRNA and typically begins at the upstream end withthe 10-nucleotide gene-start (GS) signal and ends at the downstream endwith the 12 to 13-nucleotide gene-end (GE) signal. Ten such genes foruse within the invention are known for RSV, namely NS1, NS2, N, P, M,SH, G, F, M2 and L. The term “gene” is also used herein to refer to a“translational open reading frame” (ORF). ORF is more specificallydefined as a translational open reading frame encoding a significant RSVprotein, of which 11 are currently recognized: NS1, NS2, N, P, M, SH, G,F, M2-1 (alternatively, M2(ORF1)), M2-2 (alternatively, M2(ORF2)), andL. Thus, the term “gene” interchangeably refers to a genomic RNAsequence that encodes a subgenomic RNA, and to a ORF (the latter termapplies particularly in a situation such as in the case of the RSV M2gene, where a single mRNA contains two overlapping ORFs that encodedistinct proteins). Collins et al., J. Gen. Virol. 71:3015-3020, 1990;Bermingham and Collins, Proc. Natl. Acad. Sci. USA 96:11259-11264, 1999;Ahmadian et al., EMBO J. 19:2681-2689, 2000; Jin et al., J. Virol.74:74-82, 2000 (each incorporated herein by reference). When the term“gene” is used in the context of determining gene position relative to apromoter position, the term ordinarily refers strictly to anmRNA-encoding sequence bordered by transcription gene-start and gene-endsignal motifs (Collins et al., Proc. Natl. Acad. Sci. USA 83:4594-4598,1986; Kuo et al., J. Virol. 70:6892-6901, 1996; each incorporated hereinby reference).

By “genome segment” is meant any length of continuous nucleotides fromthe RSV genome, which may be part of an ORF, a gene, or an extragenicregion, or a combination thereof.

Genes and genome segments that may be selected for use as inserts,substitutions, deleted elements, or rearranged elements within geneposition-shifted RSV of the invention include genes or genome segmentsencoding a NS1, NS2, N, P, M, SH, M2(ORF1), M2(ORF2), L, F or G proteinor portion thereof. Regulatory regions, such as the extragenic leader ortrailer regions, can also be considered. In preferred embodiments of theinvention, chimeric RSV incorporates one or more heterologous gene(s)that encode an RSV F, G or SH glycoprotein. Alternatively, therecombinant RSV may incorporate a genome segment encoding a cytoplasmicdomain, transmembrane domain, ectodomain or immunogenic epitope of a RSVF, G or SH glycoprotein. These immunogenic proteins, domains andepitopes are particularly useful within gene position-shifted RSVbecause they generate novel immune responses in an immunized host. Inparticular, the G and F proteins, and immunogenic domains and epitopestherein, provide major neutralization and protective antigens. Inaddition, genes and genome segments encoding non-RSV proteins, forexample, an SH protein as found in mumps and SV5 viruses, may beincorporated within gene position-shifted RSV of the invention.Regulatory regions, such as the extragenic 3′ leader or 5′ trailerregions, and gene-start, gene-end, intergenic regions, or 3′ or 5′non-coding regions, are also useful as heterologous (originating from adifferent RSV strain or subgroup or from a non-RSV source such as PIV,measles, mumps, etc.) substitutions or additions.

For example, addition or substitution of one or more immunogenic gene(s)or genome segment(s) from a human RSV subgroup or strain to or within abovine recipient genome or antigenome yields a recombinant, chimericvirus or subviral particle capable of generating an immune responsedirected against the human donor virus, including one or more specifichuman RSV subgroups or strains, while the bovine backbone confers anattenuated phenotype making the chimera a useful candidate for vaccinedevelopment. In one such exemplary embodiment, one or more human RSVglycoprotein genes F, SH, and/or G are added to or substituted within apartial or complete bovine genome or antigenome to yield an attenuated,infectious human-bovine chimera that elicits an anti-human RSV immuneresponse in a susceptible host. In other “chimeric” embodiments geneposition-shifted RSV incorporate a heterologous gene or genome segmentencoding an immunogenic protein, protein domain or epitope from multiplehuman RSV strains, for example two F or G proteins or immunogenicportions thereof from both RSV subgroups A and B. In yet additionalalternate embodiments a gene position-shifted RSV genome or antigenomeencodes a chimeric glycoprotein in the recombinant virus or subviralparticle having both human and bovine glycoprotein domains orimmunogenic epitopes. For example, a heterologous genome segmentencoding a glycoprotein ectodomain from a human RSV F, SH or Gglycoprotein may be joined with a genome segment encoding correspondingbovine F, SH or G glycoprotein cytoplasmic and endodomains in thebackground bovine genome or antigenome.

According to the methods of the invention, human-bovine chimeric RSV maybe constructed by substituting the heterologous gene or genome segmentfor a counterpart gene or genome segment in a partial RSV backgroundgenome or antigenome. Alternatively, the heterologous gene or genomesegment may be added as a supernumerary gene or genome segment incombination with a complete (or partial if another gene or genomesegment is deleted) RSV background genome or antigenome. For example,two human RSV G or F genes or genome segments can be included, one eachfrom RSV subgroups A and B.

Often, displacement genes or genome segments (including heterologousgenes or genome segments) are added at an intergenic position within apartial or complete RSV genome or antigenome. Alternatively, the gene orgenome segment can be placed in other noncoding regions of the genome,for example, within the 5′ or 3′ noncoding regions or in other positionswhere noncoding nucleotides occur within the partial or complete genomeor antigenome. In one aspect, noncoding regulatory regions containcis-acting signals required for efficient replication, transcription,and translation, and therefore represent target sites for modificationof these functions by introducing a displacement gene or genome segmentor other mutation as disclosed herein. In more detailed aspects of theinvention, attenuating mutations are introduced into cis-actingregulatory regions to yield, e.g., (1) a tissue specific attenuation(Gromeier et al., J. Virol. 73:958-64, 1999; Zimmermann et al., J.Virol. 71:4145-9, 1997), (2) increased sensitivity to interferon(Zimmermann et al., J. Virol. 71:4145-9, 1997), (3) temperaturesensitivity (Whitehead et al., Virology 247:232-9, 1998), (4) a generalrestriction in level of replication (Men et al., J. Virol. 70:3930-7,1996; Muster et al., Proc. Natl. Acad. Sci. USA 88:5177-5181, 1991),and/or (5) host specific restriction of replication (Cahour et al.,Virology 207:68-76, 1995). These attenuating mutations can be achievedin various ways to produce an attenuated gene position-shifted RSV ofthe invention, for example by point mutations, exchanges of sequencesbetween related viruses, or deletions.

In other alternative embodiments of the invention, gene position-shiftedRSV are provided wherein the recombinant RSV is modified by deletion,insertion substitution, or rearrangement of a plurailty of genes orgenome segments. In certain embodiments selected “gene sets” arecoordinately transferred by one of these means into, within, or from therecombinant RSV genome or antigenome. Exemplary RSV genes from whichindividual or coordinately transferred groups of genes may be selectedinclude the RSV N, P, NS1, NS2, M2-1 and M genes, which may betransferred singly or in any combination in a human or bovine RSV genomeor antigenome to yield an attenuated, gene-shifted derivative. In moredetailed aspects, both N and P genes of a human or bovine RSV aredeleted, inserted, substituted or rearranged coordinately (e.g., bycoordinate deletion or substitution in a HRSV genome or antigenome bycounterpart N and P genes from a bovine RSV). This coordinate genetransfer is facilitated by functional cooperativity between certaingenes in the RSV genome, which often arises in the case of neighboringgene pairs in the genome. Thus, in other alternative embodiments, bothNS1 and NS2 genes are coordinately transferred, e.g., by substitution ina human RSV by counterpart NS1 and NS2 genes from a bovine RSV. In yetadditional embodiments, two or more of the M2-1, M2-2 and L genes of aRSV are coordinately transferred. For certain vaccine candidates withinthe invention for which a high level of host-range restriction isdesired, each of the N, P, NS1, NS2, M2-1 and M genes of a human RSV arereplaced by counterpart N, P, NS1, NS2, M2-1 and M genes from a bovineRSV.

Coordinate gene transfers within human-bovine chimeric RSV are alsodirected to introduction of human antigenic genes within a bovinebackground genome or antigenome. In certain embodiments, one or morehuman RSV envelope-associated genes selected from F, G, SH, and M is/areadded or substituted within a partial or complete bovine RSV backgroundgenome or antigenome. For example, one or more human RSVenvelope-associated genes selected from F, G, SH, and M may be added orsubstituted within a partial bovine RSV background genome or antigenomein which one or more envelope-associated genes selected from F, G, SH,and M is/are deleted. In more detailed aspects, one or more genes from agene set defined as human RSV envelope-associated genes F, G, and M areadded within a partial bovine RSV background genome or antigenome inwhich envelope-associated genes F, G, SH, and M are deleted. Anexemplary human-bovine chimeric RSV bearing these features describe inthe examples below is rBRSV/A2-MGF.

In other aspects of the invention, insertion of heterologous nucleotidesequences into RSV vaccine candidates are employed separately tomodulate the level of attenuation of candidate vaccine recombinants,e.g., for the upper respiratory tract. Thus, it is possible to insertnucleotide sequences into a rRSV that both direct the expression of aforeign protein and that attenuate the virus in an animal host, or touse nucleotide insertions separately to attenuate candidate vaccineviruses. General tools and methods for achieving these aspects of theinvention are provided, e.g., in U.S. Provisional Patent ApplicationSer. No. 60/170,195, U.S. patent application Ser. No. 09/458,813, andU.S. patent application Ser. No. 09/459,062 (each incorporated herein byreference). In one exemplary embodiment of the invention thus provided,insertion of the measles HA ORF between a selected RSV gene junctionwill restrict viral replication in vivo. In these aspects of theinvention, the selected gene insert may be relatively large(approximately 1900 nts or greater). In this context, size of the insertspecifies a selectable level of attenuation of the resulting recombinantvirus. Displacement sequences of various lengths derived from aheterologous virus, e.g., introduced as single gene units (GUs) anddesigned specifically to lack any significant ORF, reveal selectableattenuation effects due to increased genome length (i.e., versusexpression of an additional mRNA). Other constructs in which inserts ofsimilar sizes are introduced into a downstream noncoding region (NCR) ofa RSV gene are also useful within the invention.

To define some of the rules that govern the effect of gene insertion onattenuation, gene units of varying lengths can be inserted into a wildtype RSV backbone and the effects of gene unit length on attenuationexamined. Gene unit insertions engineered to not contain a significantORF permit evaluation of the effect of gene unit length independently ofan effect of the expressed protein of that gene. These heterologoussequences were inserted in a PIV backbone as an extra gene unit of sizesbetween 168 nt and 3918 nt between the HN and L genes. In addition,control cDNA constructions and viruses were made in which insertions ofsimilar sizes were placed in the 3′-noncoding region of the HN gene ofPIV and hence did not involve the addition of an extra gene. Theseviruses were made to assess the effect of an increase in the overallgenome length and in gene number on attenuation. The insertion of anextra gene unit is expected to decrease the transcription of genesdownstream of the insertion site which will affect both the overallabundance and ratios of the expressed proteins. As demonstrated herein,gene insertions or extensions larger than about 3000 nts in lengthattenuated the wild type virus for the upper and lower respiratory tractof hamsters. Gene insertions of about 2000 nts in length furtherattenuated the rHPIV3cp45L vaccine candidate for the upper respiratorytract. Comparable gene insertions in RSV thus can have the dual effectof both attenuating a candidate vaccine virus and inducing a protectiveeffect against a second virus. Gene extensions in the 3′-noncodingregion of a gene, which cannot express additional proteins, can also beattenuating in and of themselves. Within these methods of the invention,gene insertion length is a determinant of attenuation.

A separate example of gene order rearrangement for use within theinvention involves changing the position of one or morenaturally-occurring genes relative to other naturally-occurring genes,without the introduction or deletion of substantial lengths ofpolynucleotides (e.g., greater than 100 nucleotides). For example, the Fand G genes of a human or human-bovine chimeric RSV can be shifted fromtheir natural gene order position to a more promotor proximal positionby excision and reinsertion of the genes into the recombinant genome orantigenome, without substantially altering the length of the recombinantRSV genome or antigenome.

These modifications in gene position and/or gene order typically resultin viruses with altered biological properties. For example, recombinantRSV of the invention lacking one or more selected genes, for exampleNS1, NS2, SH, or G, NS1 and NS2 together, and SH and G together, may beattenuated in vitro, in vivo, or both. Whereas this phenotype is likelyattributable primarily to the loss of expression of specific viralprotein, it is also likely that the altered gene map contributed to thephenotype. This is supported by the results observed with theSH-deletion virus, which grew more efficiently than wild type in somecell types—probably due to an increase in the efficiency oftranscription, replication or both resulting from the gene deletion andresulting change in gene order and possibly genome size.

The ability to generate infectious RSV from cDNA provides a method forintroducing predetermined changes into infectious virus via the cDNAintermediate. This method has been used to produce a series ofinfectious attenuated derivatives of wild type recombinant RSV strain A2that contain attenuating mutations including, for example, one or morenucleotide substitutions in cis-acting RNA signals and/or one or moreamino acid substitutions in one or more viral proteins and/or deletionof one or more genes or ablation its/their expression (Bukreyev et al.,J. Virol. 71:8973-8982, 1997; Whitehead et al., J. Virol. 72:4467-4471,1998; Whitehead et al., Virology 247:232-239, 1998; Bermingham andCollins, Proc. Natl. Acad. Sci. USA 96:11259-11264, 1999; Juhasz et al.,Vaccine 17:1416-1424, 1999; Juhasz et al., J. Virol. 73:5176-5180, 1999;Teng and Collins, J. Virol. 73:466-473, 1999; Whitehead et al., J.Virol. 73:871-877, 1999; Whitehead et al., J. Virol. 73:3438-3442, 1999;Collins et al., Adv. Virus Res. 54:423-451, 1999; U.S. ProvisionalPatent Application 60/143,097, filed Jul. 9, 1999, U.S. patentapplication Ser. No. 09/611,829 and its corresponding PCT applicationpublished as WO 01/04321; each incorporated herein by reference).

Strain A2 represents antigenic subgroup A, and an effective RSV vaccinealso should represent the other antigenic subgroup, subgroup B. The Gand F glycoproteins are the major antigenic determinants and the majorprotective RSV antigens (Connors et al., J. Virol. 65:1634-1637, 1991;Murphy et al., Virus Research 32:13-36, 1994; Collins et al., FieldsVirology 2:1313-1352, 1996, and Crowe et al., New Generation Vaccines,711-725, 1997; each incorporated herein by reference). Therefore, the Gand F genes of recombinant strain A2 were replaced with theircounterparts from the B1 strain of antigenic subgroup B (Whitehead etal., J. Virol. 73:9773-9780, 1999, incorporated herein by reference).This was done using wild type and attenuated strain. A2 backbones.Recombinant virus was obtained, and the “chimerization” did notdetectably interfere with virus replication. This demonstrated anexpedited method for making vaccine virus: specifically, to useattenuated strain A2 backbones to express the antigenic determinants ofsubgroup B (pending application). Thus, once an appropriately-attenuatedsubgroup A gene position-shifted RSV vaccine virus is identified inclinical trials, its backbone can be modified to produce a comparablesubgroup B vaccine in an expedited manner, and the two viruses can becombined to make a bivalent vaccine.

It has also been demonstrated in the above-incorporated references thatRSV useful within the invention can express a foreign gene added as anextra, supernumerary gene placed at any of a variety of genomiclocations, preferably in an intergenic region. This concept has beenused to make a recombinant strain A2 virus that also expressed the Gglycoprotein of subgroup B as a supernumerary gene. Thus, a single virusexpressed antigenic determinants of the two subgroups. Another exampleincorporated herein involves the expression of interferon gamma as anadded gene, which resulted in attenuation without a reduction inimmunogenicity, and also provided a method to reduce the relative levelof stimulation of T helper lymphocyte subset 2, which has been proposedto mediate immunopathogenic responses to RSV (see, e.g., U.S.Provisional Application No. 60/143,425, filed Jul. 13, 1999, U.S. patentapplication Ser. No. 09/614,285 and its corresponding PCT applicationpublished as WO 01/04271, each incorporated herein by reference).

In alternate embodiments of the invention, a different basis forattenuation of a live virus vaccine incorporating a gene positionalshift is provided, which attenuation is based in part on host rangeeffects. In this regard, the instant disclosure provides attenuated,chimeric RSV by the introduction of genome segments, entire genes ormultiple genes between HRSV and BRSV. Host range differences betweenHRSV and BRSV are exemplified by the highly permissive growth of HRSV inchimpanzees compared to the barely detectable or undetectable growth ofBRSV in the same animal. The chimpanzee is a widely accepted model ofRSV infection and immunogenic activity in humans, exhibiting virusreplication and disease comparable to that of humans. As illustratedherein below, host range differences of chimeric RSV observed inchimpanzees are correlated with host range differences observed in cellculture, providing a convenient preliminary assay.

Host range effects observed in chimeric, human-bovine RSV of theinvention are generally related to nucleotide and amino acid sequencedifferences observed between HRSV and BRSV. For example, the percentamino acid identity between HRSV and BRSV for each of the followingproteins is: NS1 (69%), NS2 (84%), N (93%), P (81%), M (89%), SH (38%),G (30%), F (81%), M2-1 (80%), L (77%). Because of the extensive geneticdivergence between HRSV and BRSV (replacement of the N gene of HRSV withthat of BRSV, for example, involves approximately 26 amino aciddifferences), chimeric bovine-human RSV of the invention areparticularly useful vaccine candidates. As exemplified herein below,replacement of the BRSV G and F glycoproteins with those of HRSVincreases the permissivity of recombinant BRSV for replication inchimpanzees. The involvement of multiple genes and genome segments eachconferring multiple amino acid or nucleotide differences provides abroad basis for attenuation which is highly stable to reversion. Thismode of attenuation contrasts sharply to HRSV viruses attenuated by oneor several point mutations, where reversion of an individual mutationwill yield a significant or complete reacquisition of virulence. Inaddition, known attenuating point mutations in HRSV typically yield atemperature sensitive phenotype. This is because the temperaturesensitive phenotype was specifically used as the first screen toidentify altered progeny following exposure of HRSV to mutagens. Oneproblem with attenuation associated with temperature sensitivity is thatthe virus can be overly restricted for replication in the lowerrespiratory tract while being under attenuated in the upper respiratorytract. This is because there is a temperature gradient within therespiratory tract, with temperature being higher (and more restrictive)in the lower respiratory tract and lower (less restrictive) in the upperrespiratory tract. The ability of an attenuated virus to replicate inthe upper respiratory tract can result in complications includingcongestion, rhinitis, fever and otitis media. Thus, attenuation achievedsolely by temperature sensitive mutations may not be ideal. In contrast,host range mutations present in gene position-shifted RSV of theinvention will not in most cases confer temperature sensitivity.Therefore, this novel method of attenuation will (i) be more stablegenetically and phenotypically, and (ii) be less likely to be associatedwith residual virulence in the upper respiratory tract than other livevaccine approaches.

The amount of sequence divergence between BRSV and HRSV is about twiceas much as between the HRSV A and B subgroups noted above. Thus, the Fproteins have approximately 20% amino acid divergence between BRSV andHRSV, and the G proteins approximately 70% divergence (Lerch, et al., J.Virol. 64:5559-69, 1990; Lerch, et al., Virology 181:118-31, 1991;Mallipeddi and Samal, J. Gen. Virol. 74:2001-4, 1993; Mallipeddi andSamal, Vet. Microbiol. 36:359-67, 1993; Samal et al., Virology180:453456, 1991; Samal and Zamora, J. Gen. Virol. 72:1717-1720, 1991;Zamora and Samal, Virus Res. 24:115-121, 1992; ibid, J. Gen. Virol.73:737-741, 1992; Mallipeddi and Samal, J. Gen. Virol. 73:2441-2444,1992, Pastey and Samal, J. Gen. Virol. 76:193-197, 1995; Walravens etal., J. Gen. Virol. 71:3009-3014, 1990; Yunnus et al., J. Gen. Virol.79:2231-2238, 1998, each incorporated herein by reference).

In the prior disclosures incorporated herein, recombinant BRSV wasmodified to replace the G and F BRSV genes with their human RSVcounterparts. The resulting chimeric BRSV/HRSV virus, bearing theantigenic determinants of human RSV on the BRSV backbone, replicatedmore efficiently in chimpanzees than did its BRSV parent, but remainedhighly attenuated. This indicated that the G and P genes contributed tothe host range restriction of BRSV, but showed that one or more othergenes also specified the host range restriction. This represents astarting point for constructing an optimal BRSV/HRSV chimeric virus thatfeatures a gene positional change as described above and which containsthe human RSV G and F antigenic determinants, wherein the resultingrecombinant RSV is attenuated by the presence of one or more BRSV genesto confer a host range restriction (Buchholz et al., J. Virol.74:1187-1199, 2000; U.S. Patent Application No. 60/143,132, filed Jul.9, 1999; each incorporated herein by reference).

Detailed descriptions of the materials and methods for producingrecombinant RSV from cDNA, and for making and testing the full range ofmutations and nucleotide modifications disclosed herein as supplementalaspects of the present invention, are set forth in, e.g., U.S.Provisional Patent Application No. 60/007,083, filed Sep. 27, 1995; U.S.patent application Ser. No. 08/720,132, filed Sep. 27, 1996; U.S.Provisional Patent Application No. 60/021,773, filed Jul. 15, 1996 andU.S. patent application Ser. No. 08/892,403, now issued as U.S. Pat. No.5,993,824; U.S. Provisional Patent Application No. 60/046,141, filed May9, 1997; U.S. Provisional Patent Application No. 60/047,634, filed May23, 1997; U.S. Pat. No. 5,993,824, issued Nov. 30, 1999 (correspondingto International Publication No. WO 98/02530); U.S. patent applicationSer. No. 09/291,894, filed by Collins et al. on Apr. 13, 1999 andcorresponding to published PCT application WO 00/61737; U.S. ProvisionalPatent Application No. 60/129,006, filed by Murphy et al. on Apr. 13,1999; Crowe et al., Vaccine 12: 691-699, 1994; and Crowe et al., Vaccine12: 783-790, 1994; Collins, et al., Proc Nat. Acad. Sci. USA92:11563-11567, 1995; Bukreyev, et al., J Virol 70:6634-41, 1996, Juhaszet al., J. Virol. 71(8):5814-5819, 1997; Durbin et al., Virology235:323-332, 1997; Karron et al., J. Infect. Dis. 176:1428-1436, 1997;He et al. Virology 237:249-260, 1997; Baron et al. J. Virol.71:1265-1271, 1997; Whitehead et al., Virology 247(2):232-9, 1998a;Whitehead et al., J. Virol. 72(5):4467-4471, 1998b; Jin et al. Virology251:206-214, 1998; Bukreyev, et al., Proc. Nat. Acad. Sci. USA96:2367-2372, 1999; Bermingham and Collins, Proc. Natl. Acad. Sci. USA96:11259-11264, 1999 Juhasz et al., Vaccine 17:1416-1424, 1999; Juhaszet al., J. Virol. 73:5176-5180, 1999; Teng and Collins, J. Virol.73:466473, 1999; Whitehead et al., J. Virol. 73:9773-9780, 1999;Whitehead et al., J. Virol. 73:871-877, 1999; and Whitehead et al., J.Virol. 73:3438-3442, 1999.

Exemplary methods for producing recombinant RSV from cDNA involveintracellular coexpression, typically from plasmids cotransfected intotissue culture cells, of an RSV antigenomic RNA and the RSV N, P, M2-1and L proteins. This launches a productive infection that results in theproduction of infectious cDNA-derived virus, which is termed recombinantvirus. Once generated, recombinant RSV is readily propagated in the samemanner as biologically-derived virus, and a recombinant virus and acounterpart biologically-derived virus cannot be distinguished unlessthe former had been modified to contain one or more introduced changesas markers.

In more detailed aspects, the foregoing incorporated documents describemethods and procedures useful within the invention for mutagenizing,isolating and characterizing RSV to obtain attenuated mutant strains(e.g., temperature sensitive (ts), cold passaged (cp) cold-adapted (ca),small plaque (sp) and host-range restricted (hr) mutant strains) and foridentifying the genetic changes that specify the attenuated phenotype.In conjunction with these methods, the foregoing documents detailprocedures for determining replication, immunogenicity, geneticstability and protective efficacy of biologically derived andrecombinantly produced attenuated human RSV, including human RSV A and Bsubgroups, in accepted model systems, including murine and non-humanprimate model systems. In addition, these documents describe generalmethods for developing and testing immunogenic compositions, includingmonovalent and bivalent vaccines, for prophylaxis and treatment of RSVinfection.

The ability to generate infectious RSV from cDNA provides a method forintroducing predetermined changes into infectious virus via the cDNAintermediate. This method has been demonstrated to produce a wide rangeof infectious, attenuated derivatives of RSV, for example recombinantvaccine candidates containing one or more amino acid substitutions in aviral protein, deletion of one or more genes or ablation of geneexpression, and/or one or more nucleotide substitutions in cis-actingRNA signals yielding desired effects on viral phenotype (see, e.g.,Bukreyev et al., J. Virol. 71:8973-8982, 1997; Whitehead et al., J.Virol. 72:44674471, 1998; Whitehead et al., Virology 247:232-239, 1998;Bermingham and Collins, Proc. Natl. Acad. Sci. USA 96:11259-11264, 1999;Juhasz et al., Vaccine 17:1416-1424, 1999; Juhasz et al., J. Virol.73:5176-5180, 1999; Teng and Collins, J. Virol. 73:466-473, 1999;Whitehead et al., J. Virol. 73:871-877, 1999; Whitehead et al., J.Virol. 73:3438-3442, 1999; and Collins et al., Adv. Virus Res.54:423-451, 1999, each incorporated herein by reference).

Exemplary of the foregoing teachings are methods for constructing andevaluating infectious recombinant RSV modified to incorporatephenotype-specific mutations identified in biologically-derived RSVmutants, e.g., cp and ts mutations adopted in recombinant RSV frombiologically derived designated cpts RSV 248 (ATCC VR 2450), cpts RSV248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), opts RSV 530(ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030(ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCCVR 2579). These methods are readily adapted for construction ofrecombinant gene position-shifted RSV of the invention. The recombinantRSV thus provided may incorporate two or more ts mutations from thesame, or different, biologically derived RSV mutant(s), for example oneor more of the 248/404, 248/955, 530/1009, or 530/1030 biologicalmutants. In the latter context, multiply attenuated recombinants mayhave a combination of attenuating mutations from two, three or morebiological mutants, e.g., a combination of attenuating mutations fromthe RSV mutants 530/1009/404, 248/404/1009, 248/404/1030, or248/404/1009/1030 mutants. In exemplary embodiments, one or moreattenuating mutations specify a temperature-sensitive substitution atamino acid Asn43, Phe521, Gln831, Met1169, or Tyr1321 in the RSVpolymerase gene or a temperature-sensitive nucleotide substitution inthe gene-start sequence of gene M2. Preferably, these mutations involveidentical or conservative changes with the following changes identifiedin biologically derived mutant RSV, for example changes conservative tothe following substitutions identified in the L polymerase gene: Ile forAsn43, Leu for Phe521, Leu for Gln831, Val for Met1169, and Asn forTyr1321.

Yet additional mutations that may be incorporated in geneposition-shifted RSV of the invention are mutations, e.g., attenuatingmutations, identified in heterologous RSV or more distantly relatednegative stranded RNA viruses. In particular, attenuating and otherdesired mutations identified in one negative stranded RNA virus may be“transferred”, e.g., copied, to a corresponding position within a humanor bovine RSV genome or antigenome, either within the geneposition-shifted RSV or as a means of constructing the geneposition-shifted RSV. Briefly, desired mutations in one heterologousnegative stranded RNA virus are transferred to the RSV recipient (e.g.,bovine or human RSV, respectively). This involves mapping the mutationin the heterologous virus, thus identifying by sequence alignment thecorresponding site in human or bovine RSV, and mutating the nativesequence in the RSV recipient to the mutant genotype (either by anidentical or conservative mutation), as described in U.S. ProvisionalPatent Application No. 60/129,006, filed by Murphy et al. on Apr. 13,1999, incorporated herein by reference. As this disclosure teaches, itis preferable to modify the chimeric genome or antigenome to encode analteration at the subject site of mutation that correspondsconservatively to the alteration identified in the heterologous mutantvirus. For example, if an amino acid substitution marks a site ofmutation in the mutant virus compared to the corresponding wild-typesequence, then a similar substitution should be engineered at thecorresponding residue(s) in the recombinant virus. Preferably thesubstitution will involve an identical or conservative amino acid to thesubstitute residue present in the mutant viral protein. However, it isalso possible to alter the native amino acid residue at the site ofmutation non-conservatively with respect to the substitute residue inthe mutant protein (e.g., by using any other amino acid to disrupt orimpair the function of the wild-type residue). Negative stranded RNAviruses from which exemplary mutations are identified and transferredinto a gene position-shifted RSV of the invention include other RSVs(e.g., murine), PIV, Sendai virus (SeV), Newcastle disease virus (NDV),simian virus 5 (SV5), measles virus (MeV), rindepest virus, caninedistemper virus (CDV), rabies virus (RaV) and vesicular stomatitis virus(VSV). A variety of exemplary mutations are disclosed, including but notlimited to an amino acid substitution of phenylalanine at position 521of the RSV L protein (corresponding to a substitution of phenylalanineat position 456 of the HPIV3 L protein). In the case of mutations markedby deletions or insertions, these can be introduced as correspondingdeletions or insertions into the recombinant virus, however theparticular size and amino acid sequence of the deleted or insertedprotein fragment can vary.

A variety of additional types of mutations are also disclosed in theforegoing incorporated references and can be readily engineered into arecombinant gene position-shifted RSV of the invention to calibrateattenuation, immunogenicity or provide other advantageous structuraland/or phenotypic effects. For example, restriction site markers areroutinely introduced within the gene position-shifted genome orantigenome to facilitate cDNA construction and manipulation. Alsodescribed in the incorporated references are a wide range of nucleotidemodifications other than point or site-specific mutations that areuseful within the instant invention. For example, methods andcompositions are disclosed for producing recombinant RSV expressing anadditional foreign gene, e.g., a chloramphenicol acetyl transferase(CAT) or luciferase gene. Such recombinants generally exhibit reducedgrowth associated with the inserted gene. This attenuation appears toincrease with increasing length of the inserted gene. The finding thatinsertion of a foreign gene into recombinant RSV reduces level ofreplication and is stable during passage in vitro provides anothereffective method for attenuating RSV for vaccine use. Similar orimproved effects can thus be achieved by insertion of other desiredgenes, for example cytokines such as interferon-γ, interleukin-2,interleukin-4 and GM-CSF, among others.

Within the methods of the invention, additional genes or genome segmentsmay be inserted into or proximate to the gene position-shifted RSVgenome or antigenome. These genes may be under common control withrecipient genes, or may be under the control of an independent set oftranscription signals. Genes of interest include the RSV genesidentified above, as well as non-RSV genes. Non-RSV genes of interestinclude those encoding cytokines (e.g., IL-2 through IL-18, especiallyIL-2, IL-6 and IL-12, IL-18, etc.), gamma-interferon, and proteins richin T helper cell epitopes. These additional proteins can be expressedeither as a separate protein, or as a chimera engineered from a secondcopy of one of the RSV proteins, such as SH. This provides the abilityto modify and improve the immune responses against RSV bothquantitatively and quantitatively.

Increased genome length results in attenuation of the resultant RSV,dependent in part upon the length of the insert. In addition, theexpression of certain proteins, e.g. a cytokine, from a non-RSV geneinserted into gene position-shifted RSV will result in attenuation ofthe virus due to the action of the protein. Exemplary cytokines thatyield an infectious, attenuated viral phenotype and high level cytokineexpression from RSV transfected cells include interleukin-2 (IL-2),IL-4, GM-CSF, and γ-interferon. Additional effects includingaugmentation of cellular and or humoral immune responses will alsoattend introduction of cytokines into gene position-shifted RSV of theinvention.

Deletions, insertions, substitutions and other mutations involvingchanges of whole viral genes or genome segments within geneposition-shifted RSV of the invention yield highly stable vaccinecandidates, which are particularly important in the case ofimmunosuppressed individuals. Many of these changes will result inattenuation of resultant vaccine strains, whereas others will specifydifferent types of desired phenotypic changes. For example, accessory(i.e., not essential for in vitro growth) genes are excellent candidatesto encode proteins that specifically interfere with host immunity (see,e.g., Kato et al., EMBO. J. 16:578-87, 1997, incorporated herein byreference). Ablation of such genes in chimeric vaccine viruses isexpected to reduce virulence and pathogenesis and/or improveimmunogenicity.

Additional, independent nucleotide modifications disclosed in theforegoing references for incorporation into recombinant geneposition-shifted RSV of the invention include partial or completedeletion or ablation of a selected RSV gene. Thus, RSV genes or genomesegments may be deleted, including partial or complete deletions of openreading frames and/or cis-acting regulatory sequences of the RSV NS1,NS2, N, P, M, G, F, SH, M2(ORF1), M2(ORF2) and/or L genes. In oneexample, a recombinant RSV was generated in which expression of the SHgene was been ablated by removal of a polynucleotide sequence encodingthe SH mRNA and protein. Deletion of the SH gene yielded not onlyrecoverable, infectious RSV, but one which exhibited substantiallyimproved growth in tissue culture based on both yield of infectiousvirus and plaque size. This improved growth in tissue culture specifiedby the SH deletion provides useful tools for developing geneposition-shifted RSV vaccines, for example by overcoming problems ofpoor RSV yields in culture. Moreover, these deletions are highly stableagainst genetic reversion, rendering RSV clones derived therefromparticularly useful as vaccine agents.

SH-minus RSV recombinants also exhibit site-specific attenuation in theupper respiratory tract of mice, which presents novel advantages forvaccine development. Current RSV strains under evaluation as live virusvaccines, for example cp mutants, do not exhibit significantly alteredgrowth in tissue culture. These are host range mutations and theyrestrict replication in the respiratory tract of chimpanzees and humansapproximately 100-fold in the lower respiratory tract. Another exemplarytype of mutation, ts mutations, tend to preferentially restrict virusreplication in the lower respiratory tract due to the gradient ofincreasing body temperature from the upper to the lower respiratorytract. In contrast to these cp and ts mutants, SH-minus RSV mutants havedistinct phenotypes of greater restriction in the upper respiratorytract. This is particularly desirable for vaccine viruses for use invery young infants, because restriction of replication in the upperrespiratory tract is required to ensure safe vaccine administration inthis vulnerable age group whose members breathe predominantly throughthe nose. Further, in any age group, reduced replication in the upperrespiratory tract will reduce morbidity from otitis media. In additionto these advantages, the nature of SH deletion mutations, involvinge.g., nearly 400 nt and ablation of an entire mRNA, represents a type ofmutation which will be highly refractory to reversion. The utility ofthe SH-minus deletion as a “displacement polynucleotide” forsimultaneously directing a gene position-shift, e.g., to upregulate Fand G glycoprotein gene expression, is amply evinced in the examplesbelow.

Also discussed in the context of SH gene modifications is a comparisonof SH genes among different RSVs, including human and bovine RSVs, andother pneumoviruses to provide additional tools and methods forgenerating useful RSV recombinant vaccines. For example, the two RSVantigenic subgroups, A and B, exhibit a relatively high degree ofconservation in certain SH domains. In two such domains, the N-terminalregion and putative membrane-spanning domains of RSV A and B display 84%identity at the amino acid level, while the C-terminal putativeectodomains are more divergent (approx. 50% identity). Comparison of theSH genes of two human RSV subgroup B strains, 8/60 and 18537, identifiedonly a single amino acid difference (Anderson et al., supra). The SHproteins of human versus bovine RSV are approximately 40% identical, andshare major structural features including (i) an asymmetric distributionof conserved residues; (ii) very similar hydrophobicity profiles; (iii)the presence of two N-linked glycosylation sites with one site being oneach side of the hydrophobic region; and (iv) a single cysteine residueon the carboxyterminal side of the central hydrophobic region of each SHprotein. (Anderson et al., supra). By evaluating these and othersequence similarities and differences, selections can be made ofheterologous sequence(s) that can be substituted or inserted withininfectious gene position-shifted RSV clones, for example to yieldvaccines having multi-specific immunogenic effects or, alternatively orin addition, desirable effects such as attenuation.

In alternate embodiments of the invention, partial gene deletions orother limited nucleotide deletions are engineered into recombinant RSVto yield desired phenotypic changes. In one example, the length of theRSV genome is reduced by deleting sequence from the downstream noncodingregion of the SH gene. This exemplary partial gene deletion wasconstructed using a version of the antigenome cDNA containing an XmaIsite in the G-F intergenic region, a change which of itself would not beexpected to affect the encoded virus. The encoded virus, designatedRSV/6120, has silent nucleotide substitutions in the last three codonsand termination codon of the SH ORF and has a deletion of 112nucleotides from the SH downstream non-translated region (positions44994610 in the recombinant antigenome). This deletion leaves thegene-end signal (Bukreyev, et al., J. Virol. 70:6634-41, 1996,incorporated herein by reference) intact. These point mutations and112-nt deletion do not alter the encoded amino acids of any of the viralproteins, nor do they interrupt any of the known viral RNA signals orchange the number of encoded mRNAs.

The 6120 virus was analyzed for the efficiency of multi-step growth inparallel with its full-length counterpart, D53, and showed a peak titerthat was reproducibly higher than that of the D53 virus by a factor of1.5- to 2-fold. Thus, the small, partial deletion in the SH gene of a112-nt noncoding sequence resulted in a substantial increase in growthefficiency in vitro.

Other partial gene deletions and small nucleotide deletions can bereadily engineered in recombinant RSV of the invention to alter viralphenotype, including nucleotide deletions in: (1) nontranslated sequenceat the beginning and/or end of the various ORFs apart from thecis-acting RNA signal, (2) intergenic regions, and (3) the regions ofthe 3′leader and 5′ trailer that are not essential for promoteractivity. Examples of nontranslated gene sequence for deletion orinsertion include the following regions of the downstream untranslatedregion of the NS1, NS2, P, M, F, and M2 genes: namely, sequencepositions 519-563, 1003-1086, 3073-3230, 4033-4197, 7387-7539 and8433-8490, respectively, numbered according to the recombinantantigenome. Also, as additional examples, nt 55-96, nt 606-624, nt42314300 can be deleted from the upstream nontranslated region of theNS1, NS2 and SH genes respectively. Any partial or complete deletion inone or more of these sequences can be achieved in accordance with theteachings herein to provide candidates that are readily screened forbeneficial phenotypic changes specified by selected deletions.Additional nontranslated regions within the RSV genome are also usefulin this regard. Since the gene-start and gene-end signals have beenmapped and characterized with regard to important positions (Kuo, etal., J. Virol., 71:49444953; 1997; Harmon, et al., J. Virol., 75:36-44,2001, each incorporated herein by reference), deletions or modificationsthat involve one or a few (e.g., 3-10, 10-20, 20-45) nt can beconsidered. In some cases, specific additional advantages may beobtained. For example, in the G gene, deletion of nt 4683 to 4685, whichincludes one nt of the gene-start signal and two nt of nontranslatedsequence, ablates the first AUG in the mRNA, which does not initiate asignificant ORF but is thought to divert ribosomes from the next AUGwhich initiates the G ORF. In addition, this deletion restores the GSsignal and retains the translation start site of the G ORF. Thus,nontranslated sites for modification can be selected based on knowledgeof the genome, or can be selected at random and tested expeditiously bythe methods of the present invention.

With regard to intergenic sequences, studies with minigenomes show thatan intergenic region can be reduced to a single nt or deleted altogetherwithout affecting transcription and RNA replication. The intergenicregions of strain A2 represent another 207 nt in aggregate (noting thatthe NS2-N intergenic region of the recombinant antigenome was engineeredto be 1 nt longer than its biological equivalent; see, e.g., Collins, etal., Proc. Natl. Acad. Sci. USA, 92:11563-11567, 1995, incorporatedherein by reference).

The RSV 5′ trailer region is 155 nt in length and thus is approximately100 nt longer than the corresponding region of most mononegaviruses andis 11 nt longer than the RSV leader region. Studies with minigenomessuggest that much of this sequence is not essential and is a candidatefor modification (Kuo, et al., J. Virol., 70:6892-901, 1996,incorporated herein by reference). For example, the region of trailerthat immediately follows the L gene could be reduced in size by 75 nt,100 nt, 125 nt or more, leaving intact the 5′ genomic terminus (whichencodes the 3′ end of the antigenome, including the antigenomepromoter). Similarly, the 44-nt leader region might be modified. Forexample, the first 11 nt at the 3′ leader end form the core of the viralpromoter, and thus sequence from the remainder of the leader regionmight be deleted or otherwise modified.

In certain embodiments of the invention, deleting one or more of thenontranslated sequences (partially or completely) described above forthe NS1, NS2, SH, F and M2 genes will result in an adjustable reductionin genome length of up to 806 nt—more than 7-fold greater than the112-nt deletion described in the instant example. Deleting partially orcompletely one or more of the intergenic regions between the first ninegenes (e.g., down to a minimal length of one nt each) would yield up toan additional 198 nt of adjustable deletion. Partial or completedeletions from the trailer and/or leader can yield up to 50, 75, 100, ormore nt in additional deletion. Thus, for example, combining 806 nt fromnontranslated gene sequence with 198 nt from the intergenic regions and100 nt from the trailer yields 1104 nt in aggregate, representing nearlya 10-fold greater deletion than 112-nt deletion described here (andrepresenting more than 7% of the RSV genome).

In another example described in the above-incorporated references,expression of the NS2 gene is ablated by introduction of stop codonsinto the translational open reading frame (ORF). The rate of release ofinfectious virus was reduced for this NS2 knock-out virus compared towild-type. In addition, comparison of the plaques of the mutant andwild-type viruses showed that those of the NS2 knock-out were greatlyreduced in size. This type of mutation can thus be incorporated withinviable recombinant gene position-shifted RSV to yield alteredphenotypes, in this case reduced rate of virus growth and reduced plaquesize in vitro. These and other knock-out methods and mutants willtherefore provide for yet additional recombinant RSV vaccine agents,based on the known correlation between reduced plaque size in vitro andattenuation in vivo. Expression of the NS2 gene also was ablated bycomplete removal of the NS2 gene, yielding a virus with a similarphenotype.

Other RSV genes which have been successfully deleted include the NS1 andM2-2 genes. The former was deleted by removal of the polynucleotidesequence encoding the respective protein, and the latter by introducinga frame-shift or altering translational start sites and introducing stopcodons. Interestingly, recovered NS1-minus virus produce small plaquesin tissue culture albeit not as small as those of the NS2 deletionvirus. The fact that the NS1-minus virus can grow, albeit with reducedefficiency, identifies the NS1 protein as an accessory protein, one thatis dispensable to irus growth. The plaque size of the NS1-minus viruswas similar to that of NS2 knock-out virus in which expression of theNS2 protein was ablated by introducing translational stop codons intoits coding sequence The small plaque phenotype is commonly associatedwith attenuating mutations. This type of mutation can thus beindependently incorporated within viable recombinant RSV to yieldaltered phenotypes. These and other knock-out methods and mutants willtherefore provide for yet additional recombinant gene position-shiftedRSV vaccine agents, based on the known correlation between plaque sizein vitro and attenuation in vivo. The NS2 knock-out mutant exhibited amoderately attenuated phenotype in the upper respiratory tract and ahighly attenuated phenotype in the lower respiratory tract in naivechimpanzees. This mutant also elicited greatly reduced disease symptomsin chimps while stimulating significant resistance to challenge by thewild-type virus (Whitehead et al., J. Virol. 73:3438-3442, 1999,incorporated herein by reference).

Yet additional methods and compositions provided within the incorporatedreferences and useful within the invention involve different nucleotidemodifications within gene position-shifted RSV that alter cis-actingregulatory sequences within the chimeric genome or antigenome. Forexample, a translational start site for a secreted form of the RSV Gglycoprotein can be deleted to disrupt expression of this form of the Gglycoprotein The RSV G protein is synthesized in two forms: as ananchored type II integral membrane protein and as a N-terminallyresected form which lacks essentially all of the membrane anchor and issecreted (Hendricks et al., J. Virol. 62:2228-2233, 1988). The two formshave been shown to be derived by translational initiation at twodifferent start sites: the longer form initiates at the first AUG of theG ORF, and the second initiates at the second AUG of the ORF at codon 48and is further processed by proteolysis (Roberts et al., J. Virol. 68:4538-4546, 1994). The presence of this second start site is highlyconserved, being present in all strains of human, bovine and ovine RSVsequenced to date. It has been suggested that the soluble form of the Gprotein might mitigate host immunity by acting as a decoy to trapneutralizing antibodies. Also, soluble G has been implicated inpreferential stimulation of a Th2-biased response, which in turn appearsto be associated with enhanced immunopathology upon subsequent exposureto RSV. With regard to an RSV vaccine virus, it is highly desirable tominimize antibody trapping or imbalanced stimulation of the immunesystem, and so it would be desirable to ablate expression of thesecreted form of the G protein. This has been achieved in recombinantvirus. Thus, this mutation is particularly useful to qualitativelyand/or quantitatively alter the host immune response elicited by therecombinant virus, rather than to directly attenuate the virus.

The incorporated references also describe modulation of the phenotype ofrecombinant RSV by altering cis-acting transcription signals ofexemplary genes, e.g., NS1 and NS2. The results of these nucleotidemodifications are consistent with modification of gene expression byaltering cis-regulatory elements, for example to decrease levels ofreadthrough mRNAs and increase expression of proteins from downstreamgenes. The resulting recombinant viruses will preferably exhibitincreased growth kinetics and increased plaque size. Exemplarymodifications to cis-acting regulatory sequences include modificationsto gene end (GE) and gene start (GS) signals associated with RSV genes.In this context, exemplary changes include alterations of the GE signalsof the NS1 and NS2 genes rendering these signals identical to thenaturally-occurring GE signals of the RSV N gene. The resultingrecombinant virus exhibits increased growth kinetics and plaque size andtherefore provide yet additional means for beneficially modifyingphenotypes of gene position-shifted RSV vaccine candidates.

Methods and compositions provided in the above-incorporated referencesalso allow production of attenuated gene position-shifted RSV vaccineviruses comprising sequences from both RSV subgroups A and B, e.g., toyield a RSV A or B vaccine or a bivalent RSV A/B vaccine (see, e.g.,U.S. patent application Ser. No. 09/291,894, filed by Collins et al. onApr. 13, 1999, incorporated herein by reference). Further augmenting theinvention in this context, specific attenuating mutations have beenincorporated into chimeric RSV A/B viruses include: (i) three of thefive cp mutations, namely the mutation in N (V267I) and the two in L(C319Y and H1690Y), but not the two in F since these are removed bysubstitution with the B1 F gene; (ii) the 248 (Q831L), 1030 (Y1321N)and, optionally, 404-L (D1183E) mutations which have been identified inattenuated strain A2 viruses; (iii) the single nucleotide substitutionat position 9 in the gene-start signal of the M2 gene, and (iv) deletionof the SH gene. Other immediately available mutations in geneposition-shifted RSV carrying RSV A and or RSV B genes or genomesegments include, but are not limited to, NS1, NS2, G, or M2-2′ genedeletions, and the 530 and 1009 mutations, alone or in combination.

In other detailed aspects of the invention, gene position-shifted RSVare employed as “vectors” for protective antigens of heterologouspathogens, including other RSVs and non-RSV viruses and non-viralpathogens. Within these aspects, the gene position-shifted RSV genome orantigenome comprises a partial or complete RSV “vector genome orantigenome” combined with one or more heterologous genes or genomesegments encoding one or more antigenic determinants of one or moreheterologous pathogens (see, e.g., U.S. Provisional Patent ApplicationSer. No. 60/170,195; U.S. patent application Ser. No. 09/458,813; andU.S. patent application Ser. No. 09/459,062, each incorporated herein byreference). The heterologous pathogen in this context may be aheterologous RSV (e.g., a different RSV strain or subgroup) and theheterologous gene(s) or genome segment(s) can be selected to encode oneor more of the above identified RSV proteins, as well as proteindomains, fragments, and immunogenic regions or epitopes thereof. RSVvector vaccines thus constructed may elicit a polyspecific immuneresponse and may be administered simultaneously or in a coordinateadministration protocol with other vaccine agents.

Gene position-shifted RSV engineered as vectors for other pathogens maycomprise a vector genome or antigenome that is a partial or completeHRSV genome or antigenome, which is combined with or is modified toincorporate one or more heterologous genes or genome segments encodingantigenic determinant(s) of one or more heterologous RSV(s), includingheterologous HRSVs selected from HRSV A or HRSV B. In alternativeaspects, the vector genome or antigenome is a partial or complete HRSVgenome or antigenome and the heterologous gene(s) or genome segment(s)encoding the antigenic determinant(s) is/are of one or more non-RSVpathogens. The vector genome or antigenome may further incorporate oneor more gene(s) or genome segment(s) of a BRSV that specifiesattenuation. Alternatively, the vector virus may be comprise a partialor complete BRSV background genome or antigenome incorporating one ormore HRSV genes or genome segments, wherein the gene position-shiftedRSV vector virus is modified to include one or more donor gene(s) orgenome segment(s) encoding an antigenic determinant of a non-RSVpathogen.

Thus, in certain detailed aspects of the invention, geneposition-shifted RSV are provided as vectors for a range of non-RSVpathogens (see, e.g., U.S. Provisional Patent Application Ser. No.60/170,195; U.S. patent application Ser. No. 09/458,813; and U.S. patentapplication Ser. No. 09/459,062, each incorporated herein by reference).The vector genome or antigenome for use within these aspects of theinvention may comprise a partial or complete BRSV or HRSV genome orantigenome incorporating, respectively, a heterologous HRSV or BRSV geneor genome segment, and the heterologous pathogen may be selected frommeasles virus, subgroup A and subgroup B respiratory syncytial viruses,HPIV1, HPIV2, BPIV3, mumps virus, human papilloma viruses, type 1 andtype 2 human immunodeficiency viruses, herpes simplex viruses,cytomegalovirus, rabies virus, Epstein Barr virus, filoviruses,bunyaviruses, flaviviruses, alphaviruses and influenza viruses.

For example, a HRSV or BRSV vector genome or antigenome for constructinggene position-shifted RSV of the invention may incorporate heterologousantigenic determinant(s) selected from the measles virus HA and Fproteins, or antigenic domains, fragments and epitopes thereof. Inexemplary embodiments, a transcription unit comprising an open readingframe (ORF) of a measles virus HA gene is added to or incorporatedwithin a BRSV or HRSV3 vector genome or antigenome. Alternatively geneposition-shifted RSV of the invention may used as vectors to incorporateheterologous antigenic determinant(s) from a parainfluenza virus (P),for example by incorporating one or more genes or genome segments thatencode(s) a HPIV1, HPIV2, or HPIV3 HN or F glycoprotein or immunogenicdomain(s) or epitope(s) thereof.

The introduction of heterologous immunogenic proteins, domains andepitopes within gene position-shifted RSV is particularly useful togenerate novel immune responses in an immunized host. For example,addition or substitution of an immunogenic gene or genome segment fromone, donor RSV subgroup or strain within a recipient genome orantigenome of a different RSV subgroup or strain can generate an immuneresponse directed against the donor subgroup or strain, the recipientsubgroup or strain, or against both the donor and recipient subgroup orstrain. To achieve this purpose, gene position-shifted RSV may also beconstructed that express a chimeric protein, e.g., an immunogenicglycoprotein having a cytoplasmic tail and/or transmembrane domainspecific to one RSV fused to an ectodomain of a different RSV toprovide, e.g., a human-bovine fusion protein, or a fusion proteinincorporating domains from two different human RSV subgroups or strains.In a preferred embodiment, a gene position-shifted RSV genome orantigenome encodes a chimeric glycoprotein in the recombinant virus orsubviral particle having both human and bovine glycoprotein domains orimmunogenic epitopes. For example, a heterologous genome segmentencoding a glycoprotein ectodomain from a human RSV F, SH or Gglycoprotein may be joined with a polynucleotide sequence (i.e., agenome segment) encoding the corresponding bovine F, SH or Gglycoprotein cytoplasmic and endo domains to form the geneposition-shifted RSV genome or antigenome.

In other embodiments, gene position-shifted RSV useful in a vaccineformulation can be conveniently modified to accommodate antigenic driftin circulating virus. Typically the modification will be in the G and/orF proteins. An entire G or F gene, or a genome segment encoding aparticular immunogenic region thereof, from one RSV strain isincorporated into a gene position-shifted RSV genome or antigenome cDNAby replacement of a corresponding region in a recipient clone of adifferent RSV strain or subgroup, or by adding one or more copies of thegene, such that several antigenic forms are represented. Progeny virusproduced from the modified RSV clone can then be used in vaccinationprotocols against emerging RSV strains.

A variety of additional embodiments of the invention involve theaddition or substitution of only a portion of a donor gene of interestto the recipient gene position-shifted RSV genome or antigenome.Commonly, non-coding nucleotides such as cis-acting regulatory elementsand intergenic sequences need not be transferred with the donor genecoding region. Thus, a coding sequence (e.g., a partial or complete openreading frame (ORF)) of a particular gene may be added or substituted tothe partial or complete background genome or antigenome under control ofa heterologous promoter (e.g., a promoter existing in the backgroundgenome or antigenome) of a counterpart gene or different gene ascompared to the donor sequence. A variety of additional genome segmentsprovide useful donor polynucleotides for inclusion within a chimericgenome or antigenome to express gene position-shifted RSV having noveland useful properties. For example, heterologous genome segments mayencode part or all of a glycoprotein cytoplasmic tail region,transmembrane domain or ectodomain, an epitopic site or region, abinding site or region containing a binding site, an active site orregion containing an active site, etc., of a selected protein from ahuman or bovine RSV. These and other genome segments can be added to acomplete background genome or antigenome or substituted therein for acounterpart genome segment to yield novel chimeric RSV recombinants.Certain recombinants will express a chimeric protein, e.g., a proteinhaving a cytoplasmic tail and/or transmembrane domain of one RSV fusedto an ectodomain of another RSV.

Genes and genome segments for use within the gene position-shifted RSVof the invention embrace an assemblage of alternate polynucleotideshaving a range of size and sequence variation. Useful genome segments inthis regard range from about 15-35 nucleotides in the case of genomesegments encoding small functional domains of proteins, e.g., epitopicsites, to about 50, 75, 100, 200-500, and 500-1,500 or more nucleotidesfor genome segments encoding larger domains or protein regions.Selection of counterpart genes and genome segments relies on sequenceidentity or linear correspondence in the genome between the subjectcounterparts. In this context, a selected human or bovine polynucleotide“reference sequence” is defined as a sequence or portion thereof presentin either the donor or recipient genome or antigenome. This referencesequence is used as a defined sequence to provide a rationale basis forsequence comparison with the counterpart heterologous sequence. Forexample, the reference sequence may be a defined a segment of a cDNA orgene, or a complete cDNA or gene sequence. Generally, a referencesequence for use in defining counterpart genes and genome segments is atleast 20 nucleotides in length, frequently at least 25 nucleotides inlength, and often at least 50 nucleotides in length. Since twopolynucleotides may each (1) comprise a sequence (i.e., a portion of thecomplete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a “comparison window” toidentify and compare local regions of sequence similarity. A “comparisonwindow”, as used herein, refers to a conceptual segment of at least 20contiguous nucleotide positions wherein a polynucleotide sequence may becompared to a reference sequence of at least 20 contiguous nucleotidesand wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of (Smith & Waterman, Adv.Appl. Math. 2:482, 1981), by the homology alignment algorithm of(Needleman & Wunsch, J. Mol. Biol. 48:443, 1970), by the search forsimilarity method of (Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988) (each of which is incorporated by reference), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package Release 7.0,Genetics Computer Group, 575 Science Dr., Madison, Wis., incorporatedherein by reference), or by inspection, and the best alignment (i.e.,resulting in the highest percentage of sequence similarity over thecomparison window) generated by the various methods is selected. Theterm “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity.

Corresponding residue positions, e.g., two different human RSVs orbetween a bovine and human RSV, may be divergent, identical or maydiffer by conservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a conservative group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Stereoisomers (e.g., D-amino acids) of the twentyconventional amino acids, unnatural amino acids such asα,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, andother unconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,ω-N-methylarginine, and other amino and imino acids (e.g.,4-hydroxyproline). Moreover, amino acids may be modified byglycosylation, phosphorylation and the like.

The present invention employs cDNA-based methods to construct a varietyof recombinant, gene position-shifted RSV viruses and subviralparticles. These recombinant RSV offer improved characteristics ofattenuation and immunogenicity for use as vaccine agents. Desiredphenotypic changes that are engineered into gene position-shifted RSVinclude, but are not limited to, attenuation in culture or in a selectedhost environment, resistance to reversion from the attenuated phenotype,enhanced immunogenic characteristics (e.g., as determined byenhancement, or diminution, of an elicited immune response),upregulation or downregulation of transcription and/or translation ofselected viral products, etc. In preferred aspects of the invention,attenuated, gene position-shifted RSV are produced in which the chimericgenome or antigenome is further modified by introducing one or moreattenuating mutations specifying an attenuating phenotype. Thesemutations may be generated de novo and tested for attenuating effectsaccording to a rational design mutagenesis strategy as described in theabove-incorporated references. Alternatively, the attenuating mutationscan be identified in a biologically derived mutant RSV and thereafterincorporated into the gene position-shifted RSV of the invention.

Attenuating mutations in biologically derived RSV for incorporationwithin a gene position-shifted RSV vaccine strain may occur naturally ormay be introduced into wild-type RSV strains by well known mutagenesisprocedures. For example, incompletely attenuated parental RSV strainscan be produced by chemical mutagenesis during virus growth in cellcultures to which a chemical mutagen has been added, by selection ofvirus that has been subjected to passage at suboptimal temperatures inorder to introduce growth restriction mutations, or by selection of amutagenized virus that produces small plaques (sp) in cell culture, asgenerally described herein and in U.S. Pat. No. 5,922,326, issued Jul.13, 1999, incorporated herein by reference.

By “biologically derived RSV” is meant any RSV not produced byrecombinant means. Thus, biologically derived RSV include naturallyoccurring RSV of all subgroups and strains, including, e.g., naturallyoccurring RSV having a wild-type genomic sequence and RSV having genomicvariations from a reference wild-type RSV sequence, e.g., RSV having amutation specifying an attenuated phenotype. Likewise, biologicallyderived RSV include RSV mutants derived from a parental RSV strain by,inter alia, artificial mutagenesis and selection procedures.

To produce a satisfactorily attenuated RSV from biologically derivedstrains, mutations are preferably introduced into a parental strainwhich has been incompletely or partially attenuated, such as the wellknown ts-1 or ts-1NG or cpRSV mutants of the A2 strain of RSV subgroupA, or derivatives or subclones thereof. Using these and other partiallyattenuated strains additional mutation(s) can be generated that furtherattenuate the strain, e.g., to a desired level of restricted replicationin a mammalian host, while retaining sufficient immunogenicity to conferprotection in vaccinees.

Partially attenuated mutants of the subgroup A or B virus can beproduced by well known methods of biologically cloning wild-type virusin an acceptable cell substrate and developing, e.g., cold-passagedmutants thereof, subjecting the virus to chemical mutagenesis to producets mutants, or selecting small plaque or similar phenotypic mutants(see, e.g., Murphy et al., International Publication WO 93/21310,incorporated herein by reference). For virus of subgroup B, anexemplary, partially attenuated parental virus is cp 23, which is amutant of the B1 strain of subgroup B.

Various known selection techniques may be combined to produce partiallyattenuated mutants from non-attenuated subgroup A or B strains which areuseful for further derivatization as described herein. Further,mutations specifying attenuated phenotypes may be introducedindividually or in combination in incompletely attenuated subgroup A orB virus to produce vaccine virus having multiple, defined attenuatingmutations that confer a desired level of attenuation and immunogenicityin vaccinees.

As noted above, production of a sufficiently attenuated biologicallyderived RSV mutant can be accomplished by several known methods. Onesuch procedure involves subjecting a partially attenuated virus topassage in cell culture at progressively lower, attenuatingtemperatures. For example, whereas wild-type virus is typicallycultivated at about 34-37° C., the partially attenuated mutants areproduced by passage in cell cultures (e.g., primary bovine kidney cells)at suboptimal temperatures, e.g., 20-26° C. Thus, the cp mutant or otherpartially attenuated strain, e.g., ts-1 or spRSV, is adapted toefficient growth at a lower temperature by passage in MRC-5 or Verocells, down to a temperature of about 20-24° C., preferably 20-22° C.This selection of mutant RSV during cold-passage substantially reducesany residual virulence in the derivative strains as compared to thepartially attenuated parent.

Alternatively, specific mutations can be introduced into biologicallyderived RSV by subjecting a partially attenuated parent virus tochemical mutagenesis, e.g., to introduce ts mutations or, in the case ofviruses which are already ts, additional ts mutations sufficient toconfer increased attenuation and/or stability of the ts phenotype of theattenuated derivative. Means for the introduction of ts mutations intoRSV include replication of the virus in the presence of a mutagen suchas 5-fluorouridine or 5-fluorouracil in a concentration of about 10⁻³ to10⁻⁵ M, preferably about 10⁻⁴ M, exposure of virus to nitrosoguanidineat a concentration of about 100 μg/ml, according to the generalprocedure described in, e.g., (Gharpure et al., J. Virol. 3:414-421,1969 and Richardson et al., J. Med. Virol. 3:91-100, 1978), or geneticintroduction of specific ts mutations. Other chemical mutagens can alsobe used. Attenuation can result from a ts mutation in almost any RSVgene, although a particularly amenable target for this purpose has beenfound to be the polymerase (L) gene.

The level of temperature sensitivity of replication in exemplaryattenuated RSV for use within the invention is determined by comparingits replication at a permissive temperature with that at severalrestrictive temperatures. The lowest temperature at which thereplication of the virus is reduced 100-fold or more in comparison withits replication at the permissive temperature is termed the shutofftemperature. In experimental animals and humans, both the replicationand virulence of RSV correlate with the mutant's shutoff temperature.Replication of mutants with a shutoff temperature of 39° C. ismoderately restricted, whereas mutants with a shutoff of 38° C.replicate less well and symptoms of illness are mainly restricted to theupper respiratory tract. A virus with a shutoff temperature of 35° C. to37° C. will typically be fully attenuated in chimpanzees andsubstantially attenuated in humans. Thus, attenuated biologicallyderived mutant and gene position-shifted RSV of the invention which arets will have a shutoff temperature in the range of about 35° C. to 39°C., and preferably from 35° C. to 38° C. The addition of a ts mutationinto a partially attenuated strain produces a multiply attenuated virususeful within vaccine compositions of the invention.

A number of attenuated RSV strains as candidate vaccines for intranasaladministration have been developed using multiple rounds of chemicalmutagenesis to introduce multiple mutations into a virus which hadalready been attenuated during cold-passage (e.g., Connors et al.,Virology 208: 478-484, 1995; Crowe et al., Vaccine 12: 691-699, 1994;and Crowe et al., Vaccine 12: 783-790, 1994, incorporated herein byreference). Evaluation in rodents, chimpanzees, adults and infantsindicate that certain of these candidate vaccine strains are relativelystable genetically, are highly immunogenic, and may be satisfactorilyattenuated. Nucleotide sequence analysis of some of these attenuatedviruses indicates that each level of increased attenuation is associatedwith specific nucleotide and amino acid substitutions. Theabove-incorporated references also disclose how to routinely distinguishbetween silent incidental mutations and those responsible for phenotypedifferences by introducing the mutations, separately and in variouscombinations, into the genome or antigenome of infectious RSV clones.This process coupled with evaluation of phenotype characteristics ofparental and derivative virus identifies mutations responsible for suchdesired characteristics as attenuation, temperature sensitivity,cold-adaptation, small plaque size, host range restriction, etc.

Mutations thus identified are compiled into a “menu” and are thenintroduced as desired, singly or in combination, to calibrate a geneposition-shifted RSV vaccine virus to an appropriate level ofattenuation, immunogenicity, genetic resistance to reversion from anattenuated phenotype, etc., as desired. Preferably, the chimeric RSV ofthe invention are attenuated by incorporation of at least one, and morepreferably two or more, attenuating mutations identified from such amenu, which may be defined as a group of known mutations within a panelof biologically derived mutant RSV strains. Preferred panels of mutantRSV strains described herein are cold passaged (cp) and/or temperaturesensitive (ts) mutants, for example a panel comprised of RSV mutantsdesignated cpts RSV 248 (ATCC VR 2450), cpts RSV 248/404 (ATCC VR 2454),cpts RSV 2481955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452), cpts RSV530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSV B-1cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCC VR 2579) (eachdeposited under the terms of the Budapest Treaty with the American TypeCulture Collection (ATCC) of 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., and granted the above identified accession numbers).

From this exemplary panel of biologically derived mutants, a large menuof attenuating mutations are provided which can each be combined withany other mutation(s) within the panel for calibrating the level ofattenuation in a recombinant, gene position-shifted RSV for vaccine use.Additional mutations may be derived from RSV having non-ts and non-cpattenuating mutations as identified, e.g., in small plaque (sp),cold-adapted (ca) or host-range restricted (hr) mutant strains.Attenuating mutations may be selected in coding portions of a donor orrecipient RSV gene or in non-coding regions such as a cis-regulatorysequence. For example, attenuating mutations may include single ormultiple base changes in a gene start sequence, as exemplified by asingle or multiple base substitution in the M2 gene start sequence atnucleotide 7605.

Gene position-shifted RSV designed and selected for vaccine use oftenhave at least two and sometimes three or more attenuating mutations toachieve a satisfactory level of attenuation for broad clinical use. Inone embodiment, at least one attenuating mutation occurs in the RSVpolymerase gene (either in the donor or recipient gene) and involves anucleotide substitution specifying an amino acid change in thepolymerase protein specifying a temperature-sensitive (ts) phenotype.Exemplary gene position-shifted RSV in this context incorporate one ormore nucleotide substitutions in the large polymerase gene L resultingin an amino acid change at amino acid Asn43, Phe521, Gln831, Met1169, orTyr1321, as exemplified by the changes, Leu for Phe521, Leu for Gln831,Val for Met1169, and Asn for Tyr1321. Alternately or additionally, geneposition-shifted RSV of the invention may incorporate a ts mutation in adifferent RSV gene, e.g., in the M2 gene. Preferably, two or morenucleotide changes are incorporated in a codon specifying an attenuatingmutation, e.g., in a codon specifying a ts mutation, thereby decreasingthe likelihood of reversion from an attenuated phenotype.

In accordance with the methods of the invention, gene position-shiftedRSV can be readily constructed and characterized that incorporate atleast one and up to a full complement of attenuating mutations presentwithin a panel of biologically derived mutant RSV strains. Thus,mutations can be assembled in any combination from a selected panel ofmutants, for example, cpts RSV 248 (ATCC VR 2450), cpts RSV 248/404(ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR2455), RSV B-1 cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCC VR2579). In this manner, attenuation of recombinant vaccine candidates canbe finely calibrated for use in one or more classes of patients,including seronegative infants.

In more specific embodiments, gene position-shifted RSV for vaccine useincorporate at least one and up to a full complement of attenuatingmutations specifying a temperature-sensitive and/or attenuating aminoacid substitution at Asn43, Phe521, Gln831, Met1169 or Tyr1321 in theRSV polymerase gene L, or a temperature-sensitive nucleotidesubstitution in the gene-start sequence of gene M2. Alternatively oradditionally, gene position-shifted RSV of claim may incorporate atleast one and up to a full complement of mutations from cold-passagedattenuated RSV, for example one or more mutations specifying an aminoacid substitution at Val267 in the RSV N gene, Glu218 or Thr523 in theRSV F gene, Cys319 or His1690 in the RSV polymerase gene L.

In other detailed embodiments, the gene position-shifted RSV of theinvention is further modified to incorporate attenuating mutationsselected from (i) a panel of mutations specifying temperature-sensitiveamino acid substitutions Gln831 to Leu, and Tyr1321 to Asn in the RSVpolymerase gene L; (ii) a temperature-sensitive nucleotide substitutionin the gene-start sequence of gene M2; (iii) an attenuating panel ofmutations adopted from cold-passaged RSV specifying amino acidsubstitutions Val267 Ile in the RSV N gene, and Cys319 Tyr and His1690Tyr in the RSV polymerase gene L; or (iv) deletion or ablation ofexpression of one or more of the RSV SH, NS1, NS2, G and M2-2 genes.Preferably, these and other examples of gene position-shifted RSVincorporate at least two attenuating mutations adopted from biologicallyderived mutant RSV, which may be derived from the same or differentbiologically derived mutant RSV strains. Also preferably, theseexemplary mutants have one or more of their attenuating mutationsstabilized by multiple nucleotide changes in a codon specifying themutation.

In accordance with the foregoing description, the ability to produceinfectious RSV from cDNA permits introduction of specific engineeredchanges within gene position-shifted RSV. In particular, infectious,recombinant RSV are employed for identification of specific mutation(s)in biologically derived, attenuated RSV strains, for example mutationswhich specify ts, ca, att and other phenotypes. Desired mutations arethus identified and introduced into recombinant, gene position-shiftedRSV vaccine strains. The capability of producing virus from cDNA allowsfor routine incorporation of these mutations, individually or in variousselected combinations, into a full-length cDNA clone, whereafter thephenotypes of rescued recombinant viruses containing the introducedmutations to be readily determined.

By identifying and incorporating specific, biologically derivedmutations associated with desired phenotypes, e.g., a cp or tsphenotype, into infectious chimeric RSV clones, the invention providesfor other, site-specific modifications at, or within close proximity to,the identified mutation. Whereas most attenuating mutations produced inbiologically derived RSV are single nucleotide changes, other “sitespecific” mutations can also be incorporated by recombinant techniquesinto biologically derived or recombinant RSV. As used herein,site-specific mutations include insertions, substitutions, deletions orrearrangements of from 1 to 3, up to about 5-15 or more alterednucleotides (e.g., altered from a wild-type RSV sequence, from asequence of a selected mutant RSV strain, or from a parent recombinantRSV clone subjected to mutagenesis). Such site-specific mutations may beincorporated at, or within the region of, a selected, biologicallyderived mutation. Alternatively, the mutations can be introduced invarious other contexts within an RSV clone, for example at or near acis-acting regulatory sequence or nucleotide sequence encoding a proteinactive site, binding site, immunogenic epitope, etc. Site-specific RSVmutants typically retain a desired attenuating phenotype, but mayadditionally exhibit altered phenotypic characteristics unrelated toattenuation, e.g., enhanced or broadened immunogenicity, and/or improvedgrowth. Further examples of desired, site-specific mutants includerecombinant RSV designed to incorporate additional, stabilizingnucleotide mutations in a codon specifying an attenuating mutation.Where possible, two or more nucleotide substitutions are introduced atcodons that specify attenuating amino acid changes in a parent mutant orrecombinant RSV clone, yielding a biologically derived or recombinantRSV having genetic resistance to reversion from an attenuated phenotype.In other embodiments, site-specific nucleotide substitutions, additions,deletions or rearrangements are introduced upstream (N-terminaldirection) or downstream (C-terminal direction), e.g., from 1 to 3, 5-10and up to 15 nucleotides or more 5′ or 3′, relative to a targetednucleotide position, e.g., to construct or ablate an existing cis-actingregulatory element.

In addition to single and multiple point mutations and site-specificmutations, changes to gene position-shifted RSV disclosed herein includedeletions, insertions, substitutions or rearrangements of whole genes orgenome segments. These mutations may alter small numbers of bases (e.g.,from 15-30 bases, up to 35-50 bases or more), large blocks ofnucleotides (e.g., 50-100, 100-300, 300-500, 500-1,000 bases), or nearlycomplete or complete genes (e.g., 1,000-1,500 nucleotides, 1,500-2,500nucleotides, 2,500-5,000, nucleotides, 5,00-6,5000 nucleotides or more)in the donor or recipient genome or antigenome, depending upon thenature of the change (i.e., a small number of bases may be changed toinsert or ablate an immunogenic epitope or change a small genomesegment, whereas large block(s) of bases are involved when genes orlarge genome segments are added, substituted, deleted or rearranged.

In alternative aspects of the invention, the infectious geneposition-shifted RSV produced from a cDNA-expressed genome or antigenomecan be any of the RSV or RSV-like strains, e.g., human, bovine, murine,etc., or of any pneumovirus, e.g., pneumonia virus of mice avianpneumovirus (previously called turkey rhinotracheitis virus). Toengender a protective immune response, the RSV strain may be one whichis endogenous to the subject being immunized, such as human RSV beingused to immunize humans. The genome or antigenome of endogenous RSV canbe modified, however, to express RSV genes or genome segments from acombination of different sources, e.g., a combination of genes or genomesegments from different RSV species, subgroups, or strains, or from anRSV and another respiratory pathogen such as PIV.

Introduction of the foregoing defined mutations into an infectious, geneposition-shifted RSV clone can be achieved by a variety of well knownmethods. By “infectious clone” with regard to DNA is meant cDNA or itsproduct, synthetic or otherwise, which can be transcribed into genomicor antigenomic RNA capable of serving as template to produce the genomeof an infectious virus or subviral particle. Thus, defined mutations canbe introduced by conventional techniques (e.g., site-directedmutagenesis) into a cDNA copy of the genome or antigenome. The use ofantigenome or genome cDNA subfragments to assemble a complete antigenomeor genome cDNA as described herein has the advantage that each regioncan be manipulated separately (smaller cDNAs are easier to manipulatethan large ones) and then readily assembled into a complete cDNA. Thus,the complete antigenome or genome cDNA, or any subfragment thereof, canbe used as template for oligonucleotide-directed mutagenesis. This canbe through the intermediate of a single-stranded phagemid form, such asusing the Muta-gene® kit of Bio-Rad Laboratories (Richmond, Calif.) or amethod using a double-stranded plasmid directly as template such as theChameleon mutagenesis kit of Stratagene (La Jolla, Calif.), or by thepolymerase chain reaction employing either an oligonucleotide primer ortemplate which contains the mutation(s) of interest. A mutatedsubfragment can then be assembled into the complete antigenome or genomecDNA. A variety of other mutagenesis techniques are known and availablefor use in producing the mutations of interest in the RSV antigenome orgenome cDNA. Mutations can vary from single nucleotide changes toreplacement of large cDNA pieces containing one or more genes or genomeregions.

Thus, in one illustrative embodiment mutations are introduced by usingthe Muta-gene phagemid in vitro mutagenesis kit available from Bio-Rad.In brief, cDNA encoding a portion of an RSV genome or antigenome iscloned into the plasmid pTZ18U, and used to transform CJ236 cells (LifeTechnologies). Phagemid preparations are prepared as recommended by themanufacturer. Oligonucleotides are designed for mutagenesis byintroduction of an altered nucleotide at the desired position of thegenome or antigenome. The plasmid containing the genetically alteredgenome or antigenome fragment is then amplified and the mutated piece isthen reintroduced into the full-length genome or antigenome clone.

The invention also provides methods for producing an infectious geneposition-shifted RSV from one or more isolated polynucleotides, e.g.,one or more cDNAs. According to the present invention cDNA encoding aRSV genome or antigenome is constructed for intracellular or in vitrocoexpression with the necessary viral proteins to form infectious RSV.By “RSV antigenome” is meant an isolated positive-sense polynucleotidemolecule which serves as the template for the synthesis of progeny RSVgenome. Preferably a cDNA is constructed which is a positive-senseversion of the RSV genome, corresponding to the replicative intermediateRNA, or antigenome, so as to minimize the possibility of hybridizingwith positive-sense transcripts of the complementing sequences thatencode proteins necessary to generate a transcribing, replicatingnucleocapsid, i.e., sequences that encode N, P, L and M2(ORF1) protein.In an RSV minigenome system, genome and antigenome were equally activein rescue, whether complemented by RSV or by plasmids, indicating thateither genome or antigenome can be used and thus the choice can be madeon methodologic or other grounds.

A native RSV genome typically comprises a negative-sense polynucleotidemolecule which, through complementary viral mRNAs, encodes elevenspecies of viral proteins, i.e., the nonstructural species NS1 and NS2,N, P, matrix (M), small hydrophobic (SH), glycoprotein (G), fusion (F),M2(ORF1), M2(ORF2), and L, substantially as described in (Mink et al.,Virology 185:615-624, 1991; Stec et al., Virology 183:273-287, 1991; andConnors et al., Virol. 208:478-484, 1995; Collins et al., Proc. Nat.Acad. Sci. USA 93:81-85, 1996), each incorporated herein by reference.For purposes of the present invention the genome or antigenome of therecombinant RSV of the invention need only contain those genes orportions thereof necessary to render the viral or subviral particlesencoded thereby infectious. Further, the genes or portions thereof maybe provided by more than one polynucleotide molecule, i.e., a gene maybe provided by complementation or the like from a separate nucleotidemolecule, or can be expressed directly from the genome or antigenomecDNA.

By recombinant RSV is meant a RSV or RSV-like viral or subviral particlederived directly or indirectly from a recombinant expression system orpropagated from virus or subviral particles produced therefrom. Therecombinant expression system will employ a recombinant expressionvector which comprises an operably linked transcriptional unitcomprising an assembly of at least a genetic element or elements havinga regulatory role in RSV gene expression, for example, a promoter, astructural or coding sequence which is transcribed into RSV RNA, andappropriate transcription initiation and termination sequences.

To produce infectious RSV from cDNA-expressed genome or antigenome, thegenome or antigenome is coexpressed with those RSV proteins necessary to(i) produce a nucleocapsid capable of RNA replication, and (ii) renderprogeny nucleocapsids competent for both RNA replication andtranscription. Transcription by the genome nucleocapsid provides theother RSV proteins and initiates a productive infection. Alternatively,additional RSV proteins needed for a productive infection can besupplied by coexpression.

An RSV antigenome may be constructed for use in the present invention byassembling cloned cDNA segments, representing in aggregate the completeantigenome, by polymerase chain reaction (PCR; described in, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202, and PCR Protocols: A Guide to Methodsand Applications, Innis et al., eds., Academic Press, San Diego, 1990,incorporated herein by reference) of reverse-transcribed copies of RSVmRNA or genome RNA. For example, cDNAs containing the lefthand end ofthe antigenome, spanning from an appropriate promoter (e.g., T7 RNApolymerase promoter) and the leader region complement to the SH gene,are assembled in an appropriate expression vector, such as a plasmid(e.g., pBR322) or various available cosmid, phage, or DNA virus vectors.The vector may be modified by mutagenesis and/or insertion of syntheticpolylinker containing unique restriction sites designed to facilitateassembly. For example, a plasmid vector described herein was derivedfrom pBR322 by replacement of the PstI-EcoR1 fragment with a syntheticDNA containing convenient restriction enzyme sites. Use of pBR322 as avector stabilized nucleotides 3716-3732 of the RSV sequence, whichotherwise sustained nucleotide deletions or insertions, and propagationof the plasmid was in bacterial strain DH10B to avoid an artifactualduplication and insertion which otherwise occurred in the vicinity of nt4499. For ease of preparation the G, F and M2 genes can be assembled ina separate vector, as can be the L and trailer sequences. The right-handend (e.g., L and trailer sequences) of the antigenome plasmid maycontain additional sequences as desired, such as a flanking ribozyme andtandem T7 transcriptional terminators. The ribozyme can be hammerheadtype (e.g., Grosfeld et al., J. Virol. 69:5677-5686, 1995), which wouldyield a 3′ end containing a single nonviral nucleotide, or can any ofthe other suitable ribozymes such as that of hepatitis delta virus(Perrotta et al., Nature 350:434436, 1991) which would yield a 3′ endfree of non-RSV nucleotides. A middle segment (e.g., G-to-M2 piece) isinserted into an appropriate restriction site of the leader-to-SHplasmid, which in turn is the recipient for theL-trailer-ribozyme-terminator piece, yielding a complete antigenome. Inan illustrative example described herein, the leader end was constructedto abut the promoter for T7 RNA polymerase which included threetranscribed G residues for optimal activity; transcription donates thesethree nonviral G's to the 5′ end of the antigenome. These three nonviralG residues can be omitted to yield a 5′ end free of nonviralnucleotides. To generate a nearly correct 3′ end, the trailer end wasconstructed to be adjacent to a hammerhead ribozyme, which upon cleavagewould donate a single 3′-phosphorylated U residue to the 3′ end of theencoded RNA.

In certain embodiments of the invention, complementing sequencesencoding proteins necessary to generate a transcribing, replicating RSVnucleocapsid are provided by one or more helper viruses. Such helperviruses can be wild-type or mutant. Preferably, the helper virus can bedistinguished phenotypically from the virus encoded by the RSV cDNA. Forexample, it is desirable to provide monoclonal antibodies which reactimmunologically with the helper virus but not the virus encoded by theRSV cDNA. Such antibodies can be neutralizing antibodies. In someembodiments, the antibodies can be used to neutralize the helper virusbackground to facilitate identification and recovery of the recombinantvirus, or in affinity chromatography to separate the helper virus fromthe recombinant virus. Mutations can be introduced into the RSV cDNAwhich render the recombinant RSV nonreactive or resistant toneutralization with such antibodies.

A variety of nucleotide insertions and deletions can be made in the geneposition-shifted RSV genome or antigenome to generate a properlyattenuated clone. The nucleotide length of the genome of wild-type humanRSV (15,222 nucleotides) is a multiple of six, and members of theParamyxovirus and Morbillivirus genera typically abide by a “rule ofsix,” i.e., genomes (or minigenomes) replicate efficiently only whentheir nucleotide length is a multiple of six (thought to be arequirement for precise spacing of nucleotide residues relative toencapsidating NP protein). Alteration of RSV genome length by singleresidue increments had no effect on the efficiency of replication, andsequence analysis of several different minigenome mutants followingpassage showed that the length differences were maintained withoutcompensatory changes. Thus, RSV lacks the strict requirement of genomelength being a multiple of six, and nucleotide insertions and deletionscan be made in the RSV genome or antigenome without defeatingreplication of the recombinant RSV of the present invention.

Alternative means to construct cDNA encoding a gene position-shifted RSVgenome or antigenome include by reverse transcription-PCR using improvedPCR conditions (e.g., as described in Cheng et al., Proc. Natl. Acad.Sci. USA 91:5695-5699, 1994; Samal et al., J. Virol. 70:5075-5082, 1996,each incorporated herein by reference) to reduce the number of subunitcDNA components to as few as one or two pieces. In other embodimentsdifferent promoters can be used (e.g., T3, SP6) or different ribozymes(e.g., that of hepatitis delta virus. Different DNA vectors (e.g.,cosmids) can be used for propagation to better accommodate the largesize genome or antigenome.

The N, P and L proteins, necessary for RNA replication, require an RNApolymerase elongation factor such as the M2(ORF1) protein for processivetranscription. Thus M2(ORF1) or a substantially equivalent transcriptionelongation factor for negative strand RNA viruses is required for theproduction of infectious RSV and is a necessary component of functionalnucleocapsids during productive infection.

The need for the M2(ORF1) protein is consistent with its role as atranscription elongation factor. The need for expression of the RNApolymerase elongation factor protein for negative strand RNA viruses isa feature of the present invention. M2(ORF1) can be supplied byexpression of the complete M2-gene, either by the chimeric genome orantigenome or by coexpression therewith, although in this form thesecond ORF2 may also be expressed and have an inhibitory effect on RNAreplication. Therefore, for production of infectious virus using thecomplete M2 gene the activities of the two ORFs should be balanced topermit sufficient expression of M2(ORF1) to provide transcriptionelongation activity yet not so much of M2(ORF2) to inhibit RNAreplication. Alternatively, the ORF1 protein is provided from a cDNAengineered to lack ORF2 or which encodes a defective ORF2. Efficiency ofvirus production may also be improved by co-expression of additionalviral protein genes, such as those encoding envelope constituents (i.e.,SH, M, G, F proteins).

In accordance with these results concerning M2(ORF2), another exemplaryembodiment of the invention is provided comprising a geneposition-shifted RSV that incorporate a mutation of M2(ORF2) (Collinsand Wertz, J. Virol. 54:65-71, 1985; Collins et al., J. Gen. Virol.71:3015-3020, 1990, Collins et al., Proc. Natl. Acad. Sci. USA 93:81-85,1996, each incorporated herein by reference) to yield novel RSV vaccinecandidates (see U.S. Provisional Patent Application No. 60/143,097,filed by Collins et al. on Jul. 9, 1999, incorporated herein byreference). In certain aspects, expression of M2 ORF2 is reduced orablated by modifying the recombinant RSV genome or antigenome toincorporate a frame shift mutation or one or stop codons in M2 ORF2yielding a “knock out” viral clone. Alternatively, M2 ORF2 is deleted inwhole or in part to render the M2-2 protein partially or entirelynon-functional or to disrupt its expression altogether to yield a M2ORF2 “deletion mutant” chimeric RSV. Alternatively, the M2-2 ORF may betranspositioned in the genome or antigenome to a more promoter-proximalor promoter-distal position compared to the natural gene order positionof M2-2 gene to up-regulate or down-regulate expression of the M2-2 ORF.In additional embodiments, the M2-2 ORF is incorporated in the genome orantigenome as a separate gene having a gene start and gene end gene endsignal, which modification results in up-regulation of the M2-2 ORF.

The gene position-shifted RSV of the invention that incorporatemutations in M2 ORF2 possess highly desirable phenotypic characteristicsfor vaccine development. The above identified modifications in therecombinant genome or antigenome specify one or more desired phenotypicchanges in the resulting virus or subviral particle. Vaccine candidatesare thus generated that exhibit one or more characteristics identifiedas (i) a change in mRNA transcription, (ii) a change in the level ofviral protein expression; (iii) a change in genomic or antigenomic RNAreplication, (iv) a change in viral growth characteristics, (v), achange in viral plaque size, and/or (vi) a change in cytopathogenicity.

In exemplary RSV recombinants incorporating an M2 ORF 2 deletion orknock out mutation, desired phenotypic changes include attenuation ofviral growth compared to growth of a corresponding wild-type or mutantparental RSV strain. In more detailed aspects, viral growth in cellculture may be attenuated by approximately 10-fold or more attributableto mutations in M2 ORF2. Kinetics of viral growth are also shown to bemodified in a manner that is beneficial for vaccine development.

Also included within the invention are M2-ORF 2 deletion and knock outmutant RSV that exhibit delayed kinetics of viral mRNA synthesiscompared to kinetics of mRNA synthesis of corresponding wild-type ormutant parental RSV strains. Despite these delayed transcriptionkinetics, these novel vaccine candidates exhibit an increase incumulative mRNA synthesis compared to parental virus. These phenotypicchanges typically are associated with an increase in viral proteinaccumulation in infected cells compared to protein accumulation in cellsinfected with wild-type or other parental RSV strains. At the same time,viral RNA replication is reduced in M2 ORF2 gene position-shifted RSVcompared to that of a parental RSV strain having normal M2 ORF2function, whereby accumulation of genomic or antigenomic RNA is reduced.

Within preferred aspects of the invention, chimeric M2 ORF2 deletion and“knock out” RSV are engineered to express undiminished or, moretypically, increased levels of viral antigen(s) while also exhibiting anattenuated phenotype. Immunogenic potential is thus preserved due to theundiminished or increased mRNA transcription and antigen expression,while attenuation is achieved through incorporation of the heterologousgene(s) or gene segment(s) and concomitant reductions in RNA replicationand virus growth attributable to the M2-ORF 2 deletion and knock outmutation. This novel suite of phenotypic traits is highly desired forvaccine development. Other useful phenotypic changes that are observedin M2 ORF2 deletion and knock out gene position-shifted RSV include alarge plaque phenotype and altered cytopathogenicity compared tocorresponding wild-type or mutant parental RSV strains.

Isolated polynucleotides (e.g., cDNA) encoding a gene position-shiftedRSV genome or antigenome and, separately, or in cis, or expressed fromthe antigenome or genome cDNA, the N, P, L and M2(ORF1) proteins, areinserted by transfection, electroporation, mechanical insertion,transduction or the like, into cells which are capable of supporting aproductive RSV infection, e.g., HEp-2, FRhL-DBS2, MRC, and Vero cells.Transfection of isolated polynucleotide sequences may be introduced intocultured cells by, for example, calcium phosphate-mediated transfection(Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic CellGenetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973),electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextranmediated transfection (Ausubel et al., (ed.) Current Protocols inMolecular Biology, John Wiley and Sons, Inc., NY, 1987, cationiclipid-mediated transfection (Hawley-Nelson et al., Focus 15:73-79, 1993)or a commercially available transfection regent, e.g., LipofectACE®(Life Technologies) (each of the foregoing references are incorporatedherein by reference).

The N, P, L and M2(ORF1) proteins are encoded by one or more cDNAs andexpression vectors which can be the same or separate from that whichencodes the genome or antigenome, and various combinations thereof.Additional proteins may be included as desired, encoded by its ownvector or by a vector encoding a N, P, L, or M2(ORF1) protein and/or thecomplete genome or antigenome. Expression of the genome or antigenomeand proteins from transfected plasmids can be achieved, for example, byeach cDNA being under the control of a promoter for T7 RNA polymerase,which in turn is supplied by infection, transfection or transductionwith an expression system for the T7 RNA polymerase, e.g., a vacciniavirus MVA strain recombinant which expresses the T7 RNA polymerase(Wyatt et al., Virology, 210:202-205, 1995, incorporated herein byreference). The viral proteins, and/or T7 RNA polymerase, can also beprovided from transformed mammalian cells, or by transfection ofpreformed mRNA or protein.

Alternatively, synthesis of antigenome or genome can be conducted invitro (cell-free) in a combined transcription-translation reaction,followed by transfection into cells. Or, antigenome or genome RNA can besynthesized in vitro and transfected into cells expressing RSV proteins.

To select candidate vaccine viruses according to the invention, thecriteria of viability, attenuation and immunogenicity are determinedaccording to well known methods. Viruses which will be most desired invaccines of the invention must maintain viability, have a stableattenuation phenotype, exhibit replication in an immunized host (albeitat lower levels), and effectively elicit production of an immuneresponse in a vaccinee sufficient to confer protection against seriousdisease caused by subsequent infection from wild-type virus. Clearly,the heretofore known and reported RS virus mutants do not meet all ofthese criteria Indeed, contrary to expectations based on the resultsreported for known attenuated RSV, viruses of the invention are not onlyviable and more appropriately attenuated than previous mutants, but aremore stable genetically in vivo than those previously studiedmutants—retaining the ability to stimulate a protective immune responseand in some instances to expand the protection afforded by multiplemodifications, e.g., induce protection against different viral strainsor subgroups, or protection by a different immunologic basis, e.g.,secretory versus serum immunoglobulins, cellular immunity, and the like.Prior to the invention, genetic instability of the ts phenotypefollowing replication in vivo has been common for ts viruses (Murphy etal., Infect. Immun. 37:235-242, 1982).

To propagate a gene position-shifted RSV virus for vaccine use and otherpurposes, a number of cell lines which allow for RSV growth may be used.RSV grows in a variety of human and animal cells. Preferred cell linesfor propagating attenuated RS virus for vaccine use include DBS-FRhL-2,MRC-5, and Vero cells. Highest virus yields are usually achieved withepithelial cell lines such as Vero cells. Cells are typically inoculatedwith virus at a multiplicity of infection ranging from about 0.001 to1.0 or more, and are cultivated under conditions permissive forreplication of the virus, e.g., at about 30-37° C. and for about 3-5days, or as long as necessary for virus to reach an adequate titer.Virus is removed from cell culture and separated from cellularcomponents, typically by well known clarification procedures, e.g.,centrifugation, and may be further purified as desired using procedureswell known to those skilled in the art.

Gene position-shifted RSV which has been attenuated and otherwisemodified as described herein can be tested in various well known andgenerally accepted in vitro and in vivo models to confirm adequateattenuation, resistance to phenotypic reversion, and immunogenicity forvaccine use. In in vitro assays, the modified virus (e.g., a multiplyattenuated, biologically derived or recombinant RSV) is tested fortemperature sensitivity of virus replication, i.e. ts phenotype, and forthe small plaque phenotype. Modified viruses are further tested inanimal models of RSV infection. A variety of animal models have beendescribed and are summarized in (Meignier et al., eds., Animal Models ofRespiratory Syncytial Virus Infection, Merieux Foundation Publication,1991, which is incorporated herein by reference). A cotton rat model ofRSV infection is described in (U.S. Pat. No. 4,800,078 and Prince etal., Virus Res. 3:193-206, 1985), which are incorporated herein byreference, and is considered predictive of attenuation and efficacy inhumans and non-human primates. In addition, a primate model of RSVinfection using the chimpanzee is predictive of attenuation and efficacyin humans, as is described in detail in (Richardson et al., J. Med.Virol. 3:91-100, 1978; Wright et al., Infect. Immun. 37:397-400, 1982;Crowe et al., Vaccine 11:1395-1404, 1993, each incorporated herein byreference).

RSV model systems, including rodents and chimpanzees for evaluatingattenuation and infectivity of RSV vaccine candidates are widelyaccepted in the art and the data obtained therefrom correlate well withRSV infection and attenuation. The mouse and cotton rat models areespecially useful in those instances in which candidate RSV virusesdisplay inadequate growth in chimpanzees, for example in the case of RSVsubgroup B viruses.

In accordance with the foregoing description and based on the examplesbelow, the invention also provides isolated, infectious geneposition-shifted RSV compositions for vaccine use. The attenuatedchimeric virus which is a component of a vaccine is in an isolated andtypically purified form. By isolated is meant to refer to RSV which isin other than a native environment of a wild-type virus, such as thenasopharynx of an infected individual. More generally, isolated is meantto include the attenuated virus as a component of a cell culture orother artificial medium where it can be propagated and characterized ina controlled setting. For example, attenuated RSV of the invention maybe produced by an infected cell culture, separated from the cell cultureand added to a stabilizer.

RSV vaccines of the invention contain as an active ingredient animmunogenically effective amount of RSV produced as described herein.Biologically derived or recombinant RSV can be used directly in vaccineformulations, or lyophilized. Lyophilized virus will typically bemaintained at about 4° C. When ready for use the lyophilized virus isreconstituted in a stabilizing solution, e.g., saline or comprising SPG,Mg++ and HEPES, with or without adjuvant, as further described below.The biologically derived or recombinantly modified virus may beintroduced into a host with a physiologically acceptable carrier and/oradjuvant. Useful carriers are well known in the art, and include, e.g.,water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid andthe like. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile solution prior to administration, as mentioned above. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents and the like, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, and the like. Acceptable adjuvants includeincomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, oralum, which are materials well known in the art. Preferred adjuvantsalso include Stimulon® QS-21 (Aquila Biopharmaceuticals, Inc.,Framingham, Mass.), MPL® (3-O-deacylated monophosphoryl lipid A; Corixa,Hamilton, Mont.), and interleukin-12 (Genetics Institute, Cambridge,Mass.).

Upon immunization with a gene position-shifted RSV vaccine compositionas described herein, via aerosol, droplet, oral, topical or other route,the immune system of the host responds to the vaccine by producingantibodies specific for one or more RSV virus proteins, e.g., F and/or Gglycoproteins. As a result of the vaccination the host becomes at leastpartially or completely immune to RSV infection, or resistant todeveloping moderate or severe RSV disease, particularly of the lowerrespiratory tract.

Gene position-shifted RSV vaccines of the invention may compriseattenuated virus that elicits an immune response against a single RSVstrain or antigenic subgroup, e.g. A or B, or against multiple RSVstrains or subgroups. In this context, the gene position-shifted RSV canelicit a monospecific immune response or a polyspecific immune responseagainst multiple RSV strains or subgroups. Alternatively, geneposition-shifted RSV having different immunogenic characteristics can becombined in a vaccine mixture or administered separately in acoordinated treatment protocol to elicit more effective protectionagainst one RSV strain, or against multiple RSV strains or subgroups.

The host to which the vaccine is administered can be any mammalsusceptible to infection by RSV or a closely related virus and capableof generating a protective immune response to antigens of thevaccinizing virus. Thus, suitable hosts include humans, non-humanprimates, bovine, equine, swine, ovine, caprine, lagamorph, rodents,etc. Accordingly, the invention provides methods for creating vaccinesfor a variety of human and veterinary uses.

The vaccine compositions containing the attenuated gene position-shiftedRSV of the invention are administered to a patient susceptible to orotherwise at risk of RSV infection in an “immunogenically effectivedose” which is sufficient to induce or enhance the individual's immuneresponse capabilities against RSV. In the case of human subjects, theattenuated virus of the invention is administered according to wellestablished human RSV vaccine protocols, as described in, e.g., (Wrightet al., Infect. Immun. 37:397400, 1982; Kim et al., Pediatrics 52:56-63,1973; and Wright et al., J. Pediatr. 88:931-936, 1976), which are eachincorporated herein by reference. Briefly, adults or children areinoculated intranasally via droplet with an immunogenically effectivedose of RSV vaccine, typically in a volume of 0.5 ml of aphysiologically acceptable diluent or carrier. This has the advantage ofsimplicity and safety compared to parenteral immunization with anon-replicating vaccine. It also provides direct stimulation of localrespiratory tract immunity, which plays a major role in resistance toRSV. Further, this mode of vaccination effectively bypasses theimmunosuppressive effects of RSV-specific maternally-derived serumantibodies, which typically are found in the very young. Also, while theparenteral administration of RSV antigens can sometimes be associatedwith immunopathologic complications, this has never been observed with alive virus.

In all subjects, the precise amount of gene position-shifted RSV vaccineadministered and the timing and repetition of administration will bedetermined based on the patient's state of health and weight, the modeof administration, the nature of the formulation, etc. Dosages willgenerally range from about 10³ to about 10⁶ plaque forming units (PFU)or more of virus per patient, more commonly from about 10⁴ to 10⁵ PFUvirus per patient. In any event, the vaccine formulations should providea quantity of attenuated RSV of the invention sufficient to effectivelystimulate or induce an anti-RSV immune response, e.g., as can bedetermined by complement fixation, plaque neutralization, and/orenzyme-linked immunosorbent assay, among other methods. In this regard,individuals are also monitored for signs and symptoms of upperrespiratory illness. As with administration to chimpanzees, theattenuated virus of the vaccine grows in the nasopharynx of vaccinees atlevels approximately 10-fold or more lower than wild-type virus, orapproximately 10-fold or more lower when compared to levels ofincompletely attenuated RSV.

In neonates and infants, multiple administration may be required toelicit sufficient levels of immunity. Administration should begin withinthe first month of life, and at intervals throughout childhood, such asat two months, six months, one year and two years, as necessary tomaintain sufficient levels of protection against native (wild-type) RSVinfection. Similarly, adults who are particularly susceptible torepeated or serious RSV infection, such as, for example, health careworkers, day care workers, family members of young children, theelderly, individuals with compromised cardiopulmonary function, mayrequire multiple immunizations to establish and/or maintain protectiveimmune responses. Levels of induced immunity can be monitored bymeasuring amounts of neutralizing secretory and serum antibodies, anddosages adjusted or vaccinations repeated as necessary to maintaindesired levels of protection. Further, different vaccine viruses may beindicated for administration to different recipient groups. For example,an engineered gene position-shifted RSV strain expressing a cytokine oran additional protein rich in T cell epitopes may be particularlyadvantageous for adults rather than for infants. RSV vaccines producedin accordance with the present invention can be combined with virusesexpressing antigens of another subgroup or strain of RSV to achieveprotection against multiple RSV subgroups or strains. Alternatively, thevaccine virus may incorporate protective epitopes of multiple RSVstrains or subgroups engineered into one RSV clone as described herein.

Typically when different vaccine viruses are used they will beadministered in an admixture simultaneously, but they may also beadministered separately. For example, as the F glycoproteins of the twoRSV subgroups differ by only about 10% in amino acid sequence, thissimilarity is the basis for a cross-protective immune response asobserved in animals immunized with RSV or F antigen and challenged witha heterologous strain. Thus, immunization with one strain may protectagainst different strains of the same or different subgroup. However,optimal protection probably will require immunization against bothsubgroups.

The gene position-shifted RSV vaccines of the invention elicitproduction of an immune response that is protective against seriouslower respiratory tract disease, such as pneumonia and bronchiolitiswhen the individual is subsequently infected with wild-type RSV. Whilethe naturally circulating virus is still capable of causing infection,particularly in the upper respiratory tract, there is a very greatlyreduced possibility of rhinitis as a result of the vaccination andpossible boosting of resistance by subsequent infection by wild-typevirus. Following vaccination, there are detectable levels of hostengendered serum and secretory antibodies which are capable ofneutralizing homologous (of the same subgroup) wild-type virus in vitroand in vivo. In many instances the host antibodies will also neutralizewild-type virus of a different, non-vaccine subgroup.

Preferred gene position-shifted RSV of the present invention exhibit avery substantial diminution of virulence when compared to wild-typevirus that is circulating naturally in humans. The gene position-shiftedvirus is sufficiently attenuated so that symptoms of infection will notoccur in most immunized individuals. In some instances the attenuatedvirus may still be capable of dissemination to unvaccinated individuals.However, its virulence is sufficiently abrogated such that severe lowerrespiratory tract infections in the vaccinated or incidental host do notoccur.

The level of attenuation of gene position-shifted RSV vaccine virus maybe determined by, for example, quantifying the amount of virus presentin the respiratory tract of an immunized host and comparing the amountto that produced by wild-type RSV or other attenuated RSV which havebeen evaluated as candidate vaccine strains. For example, the attenuatedchimeric virus of the invention will have a greater degree ofrestriction of replication in the upper respiratory tract of a highlysusceptible host, such as a chimpanzee, compared to the levels ofreplication of wild-type virus, e.g., 10- to 1000fold less. Also, thelevel of replication of the attenuated RSV vaccine strain in the upperrespiratory tract of the chimpanzee should be less than that of the RSVA2 ts-1 mutant, which was demonstrated previously to be incompletelyattenuated in seronegative human infants. In order to further reduce thedevelopment of rhinorrhea, which is associated with the replication ofvirus in the upper respiratory tract, an ideal vaccine candidate virusshould exhibit a restricted level of replication in both the upper andlower respiratory tract. However, the attenuated viruses of theinvention must be sufficiently infectious and immunogenic in humans toconfer protection in vaccinated individuals. Methods for determininglevels of RS virus in the nasopharynx of an infected host are well knownin the literature. Specimens are obtained by aspiration or washing outof nasopharyngeal secretions and virus quantified in tissue culture orother by laboratory procedure. See, for example, (Belshe et al., J. Med.Virology 1:157-162, 1977; Friedewald et al., J. Amer. Med. Assoc.204:690-694, 1968; Gharpure et al., J. Virol. 3:414-421, 1969; andWright et al., Arch. Ges. Virusforsch. 41:238-247, 1973), eachincorporated herein by reference. The virus can conveniently be measuredin the nasopharynx of host animals, such as chimpanzees.

In some instances it may be desirable to combine the geneposition-shifted RSV vaccines of the invention with vaccines whichinduce protective responses to other agents, particularly otherchildhood viruses. For example, a gene position-shifted RSV vaccine ofthe present invention can be administered simultaneously withparainfluenza virus vaccine, such as described in Clements et al., J.Clin. Microbiol. 29:1175-1182, 1991), which is incorporated herein byreference. In another aspect of the invention the chimeric RSV can beemployed as a vector for protective antigens of other respiratory tractpathogens, such as PIV, by incorporating the sequences encoding thoseprotective antigens into the gene position-shifted RSV genome orantigenome which is used to produce infectious recombinant RSV, asdescribed herein.

In yet another aspect of the invention a gene position-shifted RSV isemployed as a vector for transient gene therapy of the respiratorytract. According to this embodiment the gene position-shifted RSV genomeor antigenome incorporates a sequence which is capable of encoding agene product of interest. The gene product of interest is under controlof the same or a different promoter from that which controls RSVexpression. The infectious RSV produced by coexpressing the recombinantRSV genome or antigenome with the N, P, L and M2(ORF1) proteins andcontaining a sequence encoding the gene product of interest isadministered to a patient. This can involve a recombinant RSV which isfully infectious (i.e., competent to infect cultured cells and produceinfectious progeny), or can be a recombinant RSV which, for example,lacks one or more of the G, F and SH surface glycoprotein genes and ispropagated in cells which provide one or more of these proteins in transby stable or transient expression. In such a case, the recombinant virusproduced would be competent for efficient infection, but would be highlyinefficient in producing infectious particles. The lack of expressedcell surface glycoproteins also would reduce the efficiency of the hostimmune system in eliminating the infected cells. These features wouldincrease the durability and safety of expression of the foreign gene.

With regard to gene therapy, administration is typically by aerosol,nebulizer, or other topical application to the respiratory tract of thepatient being treated. Gene position-shifted RSV is administered in anamount sufficient to result in the expression of therapeutic orprophylactic levels of the desired gene product. Examples ofrepresentative gene products which are administered in this methodinclude those which encode, for example, those particularly suitable fortransient expression, e.g., interleukin-2, interleukin-4,gamma-interferon, GM-CSF, G-CSF, erythropoietin, and other cytokines,glucocerebrosidase, phenylalanine hydroxylase, cystic fibrosistransmembrane conductance regulator (CFTR), hypoxanthine-guaninephosphoribosyl transferase, cytotoxins, tumor suppressor genes,antisense RNAs, and vaccine antigens.

The following examples are provided by way of illustration, notlimitation.

EXAMPLE I

Construction of Recombinant RSV in which the G and F Genes Have BeenRearranged to a Promoter-Proximal Position Singly or in Combination

The present example documents rearrangement of the gene order ofinfectious RSV whereby one or more gene(s) or genome segment(s) encodingan antigenic determinant, exemplified by the G and/or F gene(s), isshifted to a more promoter-proximal position. In one example presentedbelow, the G and F genes are moved coordinately from their wild typegene order positions to occupy rearranged positions 1 and 2 of therecombinant RSV genome or antigenome. This manipulation was performedwith a cloned cDNA of RSV antigenomic RNA from which the SH gene wasdeleted (RSV ΔSH), as described above (see, e.g., Whitehead et al., J.Virol. 73:3438-3442, 1999, incorporated herein by reference). Wild typeRSV from which the SH gene is deleted grows as well or slightly betterthan complete wild type RSV in vitro, and is slightly attenuated in theupper respiratory tract of mice and in the upper and lower respiratorytracts of chimpanzee (Bukreyev et al., J. Virol. 71:8973-8982, 1997;Whitehead et al., J. Virol. 73:3438-3442, 1999, incorporated herein byreference). Its levels of immunogenicity and protective efficacy areclosely comparable to those of wild type virus. Thus, RSV ΔSH virus isslightly attenuated compared to wild type virus but otherwise has verysimilar biological properties, and it represents the parent virus inthese studies. These characteristics of the ΔSH virus, combined with theincreased genetic stability of RSV deletion mutants in general, renderthis background particularly useful as a parent recombinant forproduction of gene position-shifted RSV, facilitating recovery andmanipulation of mutant derivatives.

The antigenomic cDNA was manipulated and recombinant virus recoveredusing procedures and strategies described above (see also, Collins etal., Proc. Natl. Acad. Sci. USA 92:11563-11567, 1995; Bukreyev et al.,J. Virol. 70:6634-6641, 1996; Bukreyev et al., J. Virol. 71:8973-8982,1997; Whitehead et al., J. Virol. 72:4467-4471, 1998; Whitehead et al.,Virology 247:232-239, 1998; Bermingham and Collins, Proc. Natl. Acad.Sci. USA 96:11259-11264, 1999; Collins et al., Virology 259:251-255,1999; Bukreyev et al., Proc. Natl. Acad. Sci. USA 96:2367-2372, 1999;Juhasz et al., Vaccine 17:1416-1424, 1999; Juhasz et al., J. Virol.73:5176-5180, 1999; Teng and Collins, J. Virol. 73:466473, 1999;Whitehead et al., J. Virol. 73:9773-9780, 1999; Whitehead et al., J.Virol. 73:871-877, 1999; Whitehead et al., J. Virol. 73:3438-3442, 1999;U.S. Pat. No. 5,993,824; each incorporated herein by reference). Theantigenomic cDNA was manipulated as three subclones. One subclone,D51/ΔSH, contains the T7 promoter and left hand end of the genome fromthe leader region to the downstream end of the M gene. The secondsubclone, pUC19-GFM2, contains the G, F and M2 genes from the middle ofthe genome. The third subclone, D39, contains the L gene followed by thetrailer from the right hand end of the genome followed by aself-cleaving ribosome sequence and tandem T7 transcription terminators.

PCR with mutagenic primers was used to amplify and modify the ends ofcDNAs containing the G and F genes separately or together as a singleG-F cDNA (FIG. 1). To make a cDNA of G alone for insertion, PCR was usedto amplify nucleotides 4692-5596 of the complete recombinant RSVantigenomic sequence (spanning from the ATG that initiates the G ORF tothe downstream end of the G gene-end signal). The PCR primers weredesigned to add, immediately after the G gene-end signal, the first 6nucleotides of the G-F intergenic (IG) region followed by a copy of the10-nucleotide GS signal of the NS1 gene (FIG. 1). The PCR primers alsowere designed to add a BlpI site at both ends of the amplified cDNA. Tomake a cDNA of the F gene alone for insertion, PCR was used to amplifynucleotides 5662-7551 of the complete antigenomic sequence (spanningfrom the ATG that initiates the F ORF to the end of the F gene-endsignal). The PCR primers were designed to add, immediately after the Fgene-end signal, the first 6 nucleotides of the F-M2 IG followed by acopy of the 10-nucleotide NS1 gene-start signal. The PCR primers alsoplaced a BlpI site on both ends of the cDNA.

To make a cDNA containing the G and F genes for insertion, PCR was usedto amplify nucleotides 4692-7551 of the complete antigenomic cDNA(spanning from the ATG of the G ORF to the end of the F gene-end signal)(FIG. 1). The PCR primers were designed to add, immediately after the Fgene-end signal, the first 6 nucleotides of the F-M2 IG followed by acopy of the NS1 gene-start signal. The PCR primers also were designed toadd a BlpI site on both ends of the cDNA. All cDNAs were sequenced intheir entirety to confirm structures.

In order to make a promoter-proximal site for insertion of the G, F, orG-F cDNA, the upstream noncoding region of the NS1 gene was modified bynucleotide substitutions at antigenome positions 92 (G to C,positive-sense) and 97 (A to C), thereby creating a BlpI site (FIG. 1).This manipulation was performed on a BstBI-MfeI subclone containing thefirst 419 nucleotides of the RSV antigenomic RNA. The nucleotidesubstitutions were introduced by PCR on complete plasmid (Byrappa etal., Genome Research 5:404-407, 1995; incorporated herein by reference).The modified BstBI-MfeI fragment was reinserted into D51/ΔSH, and thissubclone served in turn as the recipient for the G, F, and G-F BlpI cDNAfragments constructed as described above. Because Blpl has an asymmetricheptameric recognition sequence, the fragments can only be inserted inthe correct orientation.

When the G, F, or G-F cDNA was placed in the promoter-proximal position,the corresponding gene(s) was/were deleted from the normal downstreamposition, so that each recombinant genome incorporated a single copy ofG and F (FIG. 1). To delete G alone, PCR was performed on pUC19-G-F-M2to amplify the StuI-HpaI fragment (nucleotides 5611-6419, spanning fromthe G-F IG into the middle of the F gene). In addition, this PCR addedthe sequence TTAATTAAAAACATATTATCACAAA (SEQ ID NO: 3) to the upstreamend of the cDNA. This sequence contains a PacI site (italicized), whichin the antigenomic cDNA is located within the SH gene-end signal. Thispiece could then be introduced as a PacI-HpaI fragment into thePacI-HpaI window of unmodified pUC19-GFM2, thereby deleting the G gene.The sequence of the cDNA fragment that had been subjected to PCR wasconfirmed by dideoxynucleotide sequencing.

Alternatively, to delete F alone, PCR was performed on pUC19-GFM2 toamplify the fragment that runs from the PacI site at nucleotide 4618 tothe G gene-end signal at nucleotide 5596. In addition, this PCR addedthe sequence CACAATTGCATGC (SEQ ID NO: 4) to the downstream end of thecDNA. This sequence contained the upstream part of the F-M2 IG sequencefollowed by an SphI site (italicized) that is present in the F-M2 IG ofthe recombinant RSV antigenome. Cloning this cDNA as a PacI-SphIfragment into the PacI-SphI window of unmodified pUC19-GFM2 resulted indeletion of the F gene. The sequence of the cDNA fragment that had beensubjected to PCR was confirmed by dideoxynucleotide sequencing.

To delete both the G and F genes, the SphI-BamHI fragment, PCR by themethod of Byrappa (Byrappa et al., Genome Research, 5:404-407 (1995)) etal. was used to amplify nucleotides 7559-8506 of pUC-GFM2 (spanning fromthe F-M2 IG to the M2/L overlap) with the following sequence added tothe upstream end of the cDNA: TTAATTAAAAACACAATT (SEQ ID NO: 5). Theresulting cDNA insert contains a PacI site (italicized) and the upstreampart of the F-M2 IG sequence that immediately precedes the SphI site,but lacks the G and, F genes. The sequence of the cDNA fragment that hadbeen subjected to PCR was confirmed by dideoxynucleotide sequencing.

Complete antigenomic cDNAs were then assembled as described above (seealso, Collins et al., Proc. Natl. Acad. Sci. USA 92:11563-11567, 1995;Bukreyev et al., J. Virol. 70:6634-6641, 1996; Bukreyev et al., J.Virol. 71:8973-8982, 1997; Whitehead et al., J. Virol. 72:4467-4471,1998; Whitehead et al., Virology 247:232-239, 1998; Bermingham andCollins, Proc. Natl. Acad. Sci. USA 96:11259-11264, 1999; Collins etal., Virology 259:251-255, 1999; Bukreyev et al., Proc. Natl. Acad. Sci.USA 96:2367-2372, 1999; Juhasz et al., Vaccine 17:1416-1424, 1999;Juhasz et al., J. Virol. 73:5176-5180, 1999; Teng and Collins, J. Virol.73:466473, 1999; Whitehead et al., J. Virol. 73:9773-9780, 1999;Whitehead et al., J. Virol. 73:871-877, 1999; Whitehead et al., J.Virol. 73:3438-3442, 1999; and U.S. Pat. No. 5,993,8244; eachincorporated herein by reference). This assembly yielded cDNAs encodingBlp/ΔSH, G1/ΔSH, F1/ΔSH, and G1F2/ΔSH antigenomic cDNAs.

These cDNAs were transfected individually into HEp-2 cells together withN, P, M2-1 and L support plasmids and incubated at 32° C. (see, e.g.,Collins et al., Proc. Natl. Acad. Sci. USA 92:11563-11567, 1995; Collinset al., Virology 259:251-255, 1999; U.S. Pat. No. 5,993,824, eachincorporated herein by reference). The transfection supernatants werepassaged to fresh cells 3 days later, and then subjected to serialpassage in HEp-2 cells at 37° C. with intervals of harvest of 3 to 7days. The shorter time intervals were necessary for monolayers infectedwith the F1/ΔSH and G1F2/ΔSH viruses because they exhibited a more rapiddevelopment of syncytia and subsequent cell destruction. This wasinterpreted to reflect increased expression of the fusogenic F proteindue to the alteration in F gene position. Aliquots of each harvestedsupernatant were flash-frozen and titrated later in parallel, as shownin FIG. 2. These results indicate that the efficiency of recovery andamplification of the G1F2/ΔSH and F1/ΔSH viruses exceeded those of theBlp/ΔSH or G1/ΔSH virus, as was also determined relative to the ΔSHvirus and wild type virus.

Total RNA was isolated from cells infected with each of the recombinantviruses, and RT-PCR of appropriate genome segments was performed whichconfirmed that the engineered genomic structures were as designed andconstructed. In addition, Northern blot analysis confirmed expression ofthe appropriate subgenomic mRNAs.

The following viruses were then compared with regard to kinetics ofgrowth and antigen production in vitro: wild type RSV (containing the SHgene), ΔSH (with the SH gene deleted but not containing a BlpI site inthe NS1 noncoding region), Blp/ΔSH, G1/ΔSH, and G1F2/ΔSH. Replicatemonolayer cultures of HEp-2 cells and Vero cells were infected at an MOIof 0.1 plaque forming units (PFU) per cell with an adsorption period of1 h (−1 to 0 hours). The monolayers were then washed three times andincubated at 37° C. Two duplicate monolayers per virus were harvested at12 h intervals, beginning immediately post-adsorption at t=0 hours. Themedium supernatants were flash-frozen for analysis later by plaque assayto quantitate released infectious virus (FIG. 3A). This showed that theviruses were comparable in their ability to produce infectious virus inVero (FIG. 3A, upper panel) and HEp-2 (FIG. 3A, lower panel) cells.

In the same experiment, the Vero cell monolayers from each time point(FIG. 3A, upper panel) were harvested and analyzed by Western blottingto characterize the expression of G protein (FIG. 4). A sample of totalinfected-cell protein from each time point was electrophoresed indenaturing polyacrylamide gel, transferred to nitrocellulose, andanalyzed by incubation with an antiserum specific to a peptide of the Gprotein. Bound antibodies were detected and quantitated using acommercial chemiluminescence kit. The antibody bound to the two majorcell-associated forms of the G protein, namely the larger 90 kDafully-mature form and the smaller 50 kDa incompletely-glycosylated form.This comparison showed that, although the production of infectiousparticles was similar for all viruses (FIG. 3A, upper panel), the amountof cell-associated G protein was considerably greater in cells infectedwith the G1/ΔSH or G1F2/ΔSH virus (FIG. 4). Quantitation of the gelbands by densitometry of the exposed film indicated that the G1/ΔSH andG1F2/ΔSH viruses expressed 6-fold and 4-fold, respectively, more Gprotein than did the Blp/ΔSH virus.

Cell monolayers infected with the F1/ΔSH or G1F2/ΔSH viruses exhibited arapid onset of cytopathic effect involving syncytium formation, asmentioned above. This was interpreted to reflect increased expression ofthe F protein. This enhanced cytopathic effect would not contraindicatethe use of this virus as a vaccine, since infection in vivo with avaccine virus involves only a small amount of epithelial cells which dieand are replaced whether the onset of cytopathogenicity for thesescattered cells is more rapid or not. However, it may be that highertiters of virus might have been achieved if the cytopathogenicity invitro had been less rapid. Resolution of these factors will be achievedby construction of gene position-shifted RSV in backgrounds that aremore highly attenuated. It is notable in this context that the geneshifts did not interfere with the growth of RSV but did increase thetotal expression of G protein by several fold.

In further studies, the ability of the gene-shift viruses to replicatein HEp-2 and Vero cell monolayers was compared in an experiment in whichthe multiplicity of infection was 3.0 (FIG. 3B). This was done in twoseparate experiments that yielded similar results. Data for one of theexperiments are shown in FIG. 3B. In this single-cycle growth assay,each of the three viruses containing G and/or F in the promoter-proximalposition, namely G1/ΔSH, F1/ΔSH, and G1F2/ΔSH, replicated moreefficiently than the control virus Blp/SH. Furthermore, the differencewas greater in Vero cells than in HEp-2 cells. For example, at 24 h postinfection, the titer of the G1/ΔSH virus was 7.2×10⁶ PFU/ml, compared to6.8×10⁶ PFU/ml for the Blp/ΔSH control in HEp-2 cells, a difference of0.4 log₁₀ whereas the respective values in Vero cells were 6.6×10⁶PFU/ml for the G1/ΔSH virus and 5.6×10⁶ PFU/ml for the Blp/ΔSH controlvirus, a difference of 1.0 log₁₀. Each of the other gene-shift virusesalso had titers in excess of that of the control virus. Thus, the shiftof G and/or F to the promoter proximal position provided, in thisexample, an increase in virus yield in both HEp-2 and Vero cells, with amaximum yield of 10-fold in Vero cells, which is a cell line that isuseful for large-scale virus production.

The gene-shift recombinant viruses also were examined for the ability toreplicate in the upper and lower respiratory tract of BALB/c mice (Table1). Mice in groups of 18 animals each were infected with 106 PFU peranimal of G1/ΔSH, F1/ΔSH, G1F2/ΔSH, or the control virus Blp/ΔSH. Sixanimals from each group were sacrificed on days 3, 4 and 5post-infection, and the nasal turbinates and lungs were harvested andanalyzed by plaque assay to determine virus titers in the upper andlower respiratory tracts, respectively. As shown in Table 1, the levelof replication of the Blp/ΔSH and F1/ΔSH viruses were essentiallyindistinguishable. Thus, although the latter virus replicated somewhatmore efficiently in vitro, its replication in vivo was not changed. Theabsence of increased replication or virulence would simplifymodification of the gene-shift viruses by the addition of attenuatingmutations. The replication of the G1F2/ΔSH virus was marginally lowerthan that of the “wild type” Blp/ΔSH control, and the replication of theG1/ΔSH virus also was lower, particularly early in the infection. Forexample, on day 3 the G1/ΔSH virus was 0.7 log₁₀ lower in both the upperand lower respiratory tract compared to the Blp/ΔSH control. TABLE 1Replication, in the upper and lower respiratory tract of mice, ofrecombinant RSV containing the G and/or F genes in the promoter-proximalposition. Nasal Turbinates mean titer ± Lungs Mean titer ± SE SE (log₁₀PFU/g tissue) (log₁₀ PFU/g tissue) Virus¹ Day 3 Day 4 Day 5 Day 3 Day 4Day 5 B1p/ΔSH 4.2 ± 0.17 4.0 ± 0.32 3.5 ± 0.2  3.6 ± 0.26 4.1 ± 0.37 4.5± 0.09 G1/ΔSH 3.5 ± 0.24 3.4 ± 0.54 3.5 ± 0.24 2.9 ± 0.2  3.9 ± 0.34 4.6± 0.13 F1/ΔSH 4.4 ± 0.11 4.1 ± 0.1  3.9 ± 0.12 3.9 ± 0.09 4.7 ± 0.08 4.9± 0.16 G1F2/ΔSH 3.3 ± 0.50 3.5 ± 0.13 3.3 ± 0.13 3.1 ± 0.13 4.2 ± 0.284.1 ± 0.071. BALB/C mice in groups of 18 were inoculated intranasally with 10⁶ PFuper mouse of the indicated virus on day 0. On days 3, 4, and 5, six miceper group were sacrificed and the nasal turbinates and lungs wereharvested and virus titers were determined by plaque assay. Mean titersare shown with the standard error indicated.

Thus, shifting one or more genes to the promoter proximal position, orrearranging RSV genes in general, can modestly attenuate the virus forreplication in vivo. This has value for designing an attenuated vaccinestrain, since it adds to the menu of useful methods of attenuation.Furthermore, it is important to have attenuating mutations thatrepresent different classes or types, such as temperature-sensitivepoint mutations, non-temperature-sensitive point mutations, genedeletions, and so forth. Different types of mutation operate indifferent ways to affect viral phenotype, and the presence of multipletypes of mutations in a single vaccine virus confers increasedstability. Attenuation by gene shift represents an additional usefulclass of attenuating mutations in this context. The finding that geneshifts according to the invention can confer a modest degree ofattenuation provides useful new tools for fine-tuning the attenuationphenotype of vaccine strains.

Certain useful attenuating mutations within the invention are of a“conditional” variety, where attenuation is minimal under specifiedconditions (e.g., in vitro) and maximal under other conditions (e.g., invivo). An attenuation phenotype that is minimal or not operant in vitropermits efficient production of vaccine virus, which is particularlyimportant in the case of RSV and other viruses that grow poorly in cellculture. Of course, as illustrated here, the attenuation phenotype mustbe operant in vivo in order to reduce disease and reactogenicity of thevaccine virus.

The G1/ΔSH recombinant shown here illustrates a particularly desirablecombination of traits resulting from the gene-shift. Specifically, itsgrowth in vitro actually was increased up to 10-fold, while itsreplication in vivo was decreased moderately. This provides theadvantage of improved efficiency of vaccine production in conjunctionwith attenuation in vivo, and improved antigen expression, as describedabove.

The immunogenicity of the gene-shift viruses in vivo was investigated inBALB/c mice. Mice in groups of six were infected with the individualviruses as described immediately above, and serum samples were taken 1day prior to inoculation and 28 and 56 days post inoculation (Table 2).The serum samples were analyzed by glycoprotein-specific enzyme-linkedimmunoadsorbent assay (ELISA) specific to IgG (Table 2). Analysis of Gprotein-specific IgG showed that the responses to the F1/ΔSH andG1F2/ΔSH viruses were very similar to that for the Blp/ΔSH controlvirus. On the other hand, the G-specific response to the G1/ΔSH virusmoderately decreased (up to four-fold). This likely is due at least inpart to the reduced replication of this virus, as described above inTable 1. Analysis of F-specific responses showed that the F1/ΔSH andG1F2/ΔSH viruses had moderate increases (2.5- to 4-fold) in antibodylevels compared to the G1/ΔSH and Blp/ΔSH viruses, which is consistentwith the interpretation that moving the F gene to the promoter-proximalposition results in increased antigen expression in vivo and increasedimmunogenicity. Furthermore, these results indicated that the gene-shiftcan result in increased immunogenicity in vivo under conditions whereoverall replication of the immunizing virus is not changed. This is ahighly desirable outcome, since it provides a specific method to make anRSV vaccine that is Δis inherently more immunogenic than the wild typeparent virus. It should be noted that the mouse model can be used toidentify and characterize biological properties of a virus and canreveal desirable new features such as shown here. TABLE 2 Measurement,by glycoprotein-specific enzyme-linked immunoabsorbent assay (ELISA), orserum antibody responses in mice following infection with recombinantRSV containing G and/or F gene in the promoter-proximal position. Serumimmunoglobulin G ELISA titer (mean reciprocal log₂ ± SE) againstindicated RSV protein² No. of Anti RSV Anti RSV animals G IgG F IgGVirus¹ per group Pre Day 28 Day 56 Pre Day 28 Day 56 B1p/ΔSH 6 ≦5.3 ± 09.6 ± 0.6 11.7 ± 0.8 ≦5.3 ± 0 10.3 ± 0.4 12.0 ± 0.4 G1/ΔSH 6 ≦5.3 ± 09.0 ± 0.6  9.6 ± 0.6 ≦5.3 ± 0 10.0 ± 0.4 12.0 ± 0.7 F1/ΔSH 6 ≦5.3 ± 09.6 ± 0.9 11.6 ± 0.6 ≦5.3 ± 0 11.6 ± 0.3 13.6 ± 0.3 G1F2/ΔSH 6 ≦5.3 ± 09.3 ± 0.7 11.6 ± 0.8 ≦5.3 ± 0 12.3 ± 0.4 14.0 ± 0.41. BALB/C mice in groups of six were inoculated intranasally with 10⁶PFU of virus in a 0.1 ml inoculum on day 0.

2. Serum samples were taken 1 day prior to inoculation (Pre) and 28 and56 days post inoculation, and were analyzed by glycoprotein-specificELISA for IgG antibodies against the RSV G or F protein, as indicated.Mean titers are shown with standard error indicated.

EXAMPLE II

Recombinant RSV Incorporating the G and F Genes in a Promoter-ProximalShifted Position in a Highly Attenuated Background

The gene shift mutations described above were introduced in the contextof a genetic background from which the SH gene was deleted. In wild typevirus, this deletion modestly improves growth in cell culture, and ismoderately attenuating in vivo (Bukreyev et al., J. Virol. 71:8973-8982,1997; whitehead et al., J. Virol. 73:3438-3442, 1999, incorporatedherein by reference). However, an RSV vaccine virus that is safe foradministration to RSV-naive infants and children requires furtherattenuation than is provided by the ΔSH deletion alone. Therefore, thepresent example is provided to demonstrate that gene shift mutants ofthe invention can be recovered successfully in a highly attenuatedbackground.

Two backgrounds were chosen to exemplify this aspect of the invention,one lacking both the SH and NS2 genes and one lacking the SH, NS1 andNS2 genes. As described in the above-incorporated references, the ΔNS2and ΔNS1 deletions are each highly attenuating on its own (see, e.g.,Whitehead et al., J. Virol. 73:3438-3442, 1999). In vitro, theproduction of virus containing either mutation is delayed and reduced,although under vaccine production conditions a yield of ΔNS2 virus isachieved comparable to that of wild type virus. In chimpanzees, the ΔNS2and ΔNS1 viruses each are highly attenuated for replication and diseaseand are highly immunogenic and protective against RSV challenge. Eachmutation alone is an excellent candidate to be included in a recombinantvaccine virus, either on its own or in combination with other mutations.The ΔNS1 and ΔNS2 mutations together result in a virus that is even morehighly attenuated in vitro than the ΔNS2 virus. In the present example,further combinations of these deletions were constructed and tested fortheir ability to support further gene position-shifted mutations.

Antigenomic cDNAs were constructed in which the G and F genes were movedto positions 1 and 2 of an antigenome from which the NS2 and SH geneshad been deleted, designated G1F2/ΔNS2ΔSH (FIG. 5, panel A), or topositions 1 and 2 of an antigenome in which the NS1, NS2 and SH geneswere deleted, designated G1F2/ΔNS1ΔNS2ΔSH (FIG. 5, panel B). Theseantigenomic cDNAs were used to recover recombinant virus as described inExample I above. In both cases, recombinant virus was readily recoveredand propagated in vitro. Thus, the present example demonstrates thatgene position-shifted RSVs containing multiple attenuating mutationswith the G and F genes shifted to the promoter-proximal positions can bereadily produced and recovered. These and other gene position-shiftedRSVs will be analyzed for levels of replication and antigen expression,as well as growth, immunogenicity and protective efficacy in vivo toselect suitable vaccine candidates in accordance with the methodsdescribed herein.

In the foregoing examples, representative changes were made in the geneorder of RSV to improve its properties as a live-attenuated vaccine. Inparticular, the G and F genes were moved, singly and in tandem, to amore promoter-proximal position. These two proteins normally occupypositions 7 (G) and 8 (F) in the RSV gene order(NS1-NS2-N-P-M-SH-G-F-M2-L). In order to increase the possibility ofsuccessful recovery, the manipulations were performed in a version ofRSV in which the SH gene had been deleted. G and F were then movedindividually to position 1, or were moved together to positions 1 and 2,respectively. Surprisingly, recombinant RSV were readily recovered inwhich G or F were moved to position 1, or in which G and F were moved topositions 1 and 2, respectively. This result differed greatly fromprevious studies with VSV, where movement of the single VSV glycoproteingene by only two positions was very deleterious to virus growth. Theability to recover these altered viruses also was surprising because RSVreplicates inefficiently and because RSV has a complex gene order andmovement of the glycoprotein genes involved a large number of positionchanges. Indeed, the rearranged RSV's grow at least as well as doestheir immediate parent having the wild type order of genes. As indicatedabove, this is particularly important for RSV, since the wild type virusgrows inefficiently in cell culture and a further reduction inreplication in vitro would likely render vaccine preparation unfeasible.It is remarkable that all of the NS1-NS2-N-P-M proteins could bedisplaced by one or two positions relative to the promoter without asignificant decrease in growth fitness. In addition, examination of theexpression of the G glycoprotein showed that it was increased up toseveral-fold over that of its parent virus. This indicated that avaccine virus containing G and/or F in the first position expresses ahigher molar amount of these protective antigens compared to the otherviral proteins, and thus represent a virus with highly desirable vaccineproperties.

Furthermore, the modification in gene order also was achieved with twohighly attenuated vaccine candidates produced in previous work, in whichthe NS2 gene was deleted on its own as described previously, or in whichthe NS1 and NS2 genes were deleted together. In these two vaccinecandidates, the G and F glycoproteins were moved together to positions 1and 2 respectively, and the G, F and SH glycoproteins were deleted fromtheir original downstream position. Thus, the recovered virusesG1F2ΔNS2ΔSH and G1F2/ΔNS1ΔNS2ΔSH had two and three genes deletedrespectively in addition to the shift of the G and F genes. Toillustrate the extent of the changes involved, the gene orders of wildtype RSV (NS1-NS2-N-P-M-SH-G-F-M2-L) and the G1F2/ΔNS2ΔSH virus(G-F-NS1-N-P-M-M2-L) or the ΔNS1ΔNS2ΔSH (G-F-N-P-M-M2-L) can becompared. This shows that the positions of most or all of the genesrelative to the promoter were changed. Nonetheless, these highlyattenuated derivatives retained the capacity to be grown in cellculture, indicating their clear utility for development of candidatevaccine viruses.

EXAMPLE III

Construction of a Chimeric BRSV/HRSV Containing the HRSV G and F Genesin a Promoter-Proximal Shifted Position

The present example describes construction of an infectious rBRSV/HRSVchimera in which the HRSV G and F genes are substituted into arecombinant bovine RSV (rBRSV) background. The resulting human-bovinechimera contains two genes of HRSV, namely G and F, and eight genes fromBRSV, namely NS1, NS2, N, P, M, SH, M2 and L. Additional detaildescribing a human-bovine RSV construct having the human G and F genessubstituted at their corresponding, wild type positions in a bovine RSVbackground (designated rBRSV/A2) is provided in U.S. patent applicationSer. No. 09/602,212, filed by Bucholz et al. on Jun. 23, 2000, itscorresponding PCT application published as WO 01/04335 on Jan. 18, 2001,and its priority provisional U.S. Application No. 60/143,132 filed onJul. 9, 1999, each incorporated herein by reference.

In addition to the basic substituted glycoprotein construction ofrBRSV/A2, the HRSV G and F genes were shifted in the present example toa more promoter-proximal position in the rBRSV backbone relative to awild type gene order position of the F and G genes in the BRSV genome.More specifically, the F and G genes were moved from their usuallocation relative to the promoter, namely gene positions 7 and 8,respectively, to positions 1 and 2, respectively. To achieve thisobjective, complete infectious rBRSV was constructed in which nucleotidesubstitutions were made to create unique NotI, SalI and XhoI sites atpositions 67, 4,673 and 7,471, respectively (FIG. 6, panel A) (see also,Buchholz et al., J. Virol. 73:251-259, 1999; Buchholz et al., J. Virol.74:1187-1199, 2000, incorporated herein by reference). The NotI site iscontained within the upstream nontranslated region of the BRSV NS1 gene,and the SalI and XhoI sites are in intergenic regions. Digestion of therBRSV antigenomic cDNA with SalI and XhoI excised the BRSV G and F genesin their entirety and created compatible cohesive ends that were ligated(FIG. 6, panel B). This resulted in an rBRSV antigenomic cDNA lackingthe G and F genes and containing a 64-nucleotide SH-M2 intergenic regionwith the following sequence:

-   TTAAACTTAAAAATGGTTTATGtcgaGGAATAAAATCGATTAACAACCAATCAT TCAAAAAGAT    (SEQ ID NO: 6) (the tetranucleotide cohesive ends of the original    cleaved SalI and XhoI sites are in small case). For comparison, the    naturally-occurring BRSV F-M2 intergenic sequence is 55 nucleotides    in length.

A cDNA containing the HRSV G and F genes was prepared by PCR withmutagenic primers used to modify the cDNA ends. Specifically, PCR wasused to amplify nucleotides 4692-7551 of the complete HRSV antigenomiccDNA (spanning from the ATG of the G ORF to the end of the F gene-endsignal), and the primers were designed to add, immediately after the Fgene-end signal, the first 6 nucleotides of the F-M2 IG followed by acopy of the NS1 gene-start signal. The PCR primers also were designed toadd a BlpI site and an NotI site each on both ends of the cDNA. Thesequence of the cDNA fragment that was subjected to PCR was confirmed bydideoxynucleotide sequencing. This cDNA was then inserted as a NotIfragment into the unique NotI site of the rBRSV antigenomic cDNA lackingthe G and F genes as described above. A correct recombinant wasidentified by restriction fragment mapping, and was designatedrBRSV/A2-G1F2. The structure of the encoded genomic RNA is shown in FIG.6, panel C. As shown, in this cDNA the G and F genes were moved frompositions 7 and 8 relative to the promoter to positions 1 and 2.

A plasmid encoding the antigenomic RNA of rBRSV/A2-G1 F2 wastransfected, together with plasmids encoding the N, P, M2-1 and Lsupport proteins, into BSR T7/5 cells, which stably express the T7 RNApolymerase, as described above (see also, Buchholz et al., J. Virol.73:251-259, 1999; Buchholz et al., J. Virol. 74:1187-1199, 2000, eachincorporated herein by reference), and infectious virus was recovered.The recovered rBRSV/A2-G1F2 virus was compared to rBRSV, rHRSV (alsocalled rA2) and rBRSV/A2 with regard to the efficiency of multicyclegrowth in human HEp-2 cells and bovine MDBK cells. As described above(see also, Buchholz et al, J. Virol. 74:1187-1199, 2000, incorporatedherein by reference), rHRSV grows much more efficiently than rBRSV inHEp-2 cells, and the rBRSV/A2 virus grows with an efficiencyintermediate between that of each parent. As shown in FIG. 7, theefficiency of replication of rBRSV/A2-G1F2 was indistinguishable fromthat of rBRSV/A2. Thus, unexpectedly, the change in the location of theG and F genes did not reduce the efficiency of growth in vitro, whichnovel result allows for efficient production of RSV vaccine virus.

Immunofluorescence was performed on HEp-2 cells infected with wt HRSV orthe chimeric viruses rBRSV/A2 or rBRSV/A2-G1F2. This was performed usingtwo monoclonal antibodies specific to the HRSV G or F proteins, namely021/01G and 44F, respectively (Lopez et al., J. Virol. 72:6922-6928,1998; Melero et al., J. Gen. Virol. 78:2411-2418, 1997, eachincorporated herein by reference). The staining was done with eachmonoclonal antibody individually. Although this assay is onlysemi-quantitative, it has been previously determined that the assaydistinguishes reliably between wt rBRSV and rBRSV/A2 (the latter bearingthe HRSV G and F genes in the normal genome location in the rBRSVbackbone). In particular, wt HRSV gives a very strong, extensive patternof immunofluorescence indicative of efficient and extensive antigenexpression, while rBRSV/A2 gives a weaker, more diffuse, less extensivepattern (Buchholz et al., J. Virol. 74:1187-1199, 2000, incorporatedherein by reference). A comparable assay conducted for rBRSV/A2-G1F2(FIG. 8), shows that the pattern of immunofluorescence for thispromoter-shifted chimeric virus was very similar to that of wt BRSV.This result is consistent with increased expression of the G and Fglycoproteins. At the same time, the cytopathic effect associated withrBRSV/A2-G1F2 was reduced compared to wt HRSV, and more closelyresembled that of rBRSV/A2 (Buchholz et al., J. Virol. 74:1187-1199,2000, incorporated herein by reference). Specifically, rBRSV/A2 andrBRSV/A2-G1F2 induced fewer and smaller syncytia.

Thus, the present example documents modification of the rBRSV/A2human-bovine chimeric RSV virus, which contains the genes for the majorprotective antigens of HRSV, the G and F proteins, in the background ofBRSV which is strongly attenuated for replication in the respiratorytract of primates. The rBRSV/A2 virus has a strong host rangerestriction that renders it highly attenuated in primates. Since thepresent gene position-shifted rBRSV/A2-G1F2 virus bears the sameconstellation of BRSV genes in its genetic background, it is likely toshare this strong host range restriction phenotype, thereby increasingthe expression of the two major protective antigens. The increasedexpression of these two protective antigens in vivo is further expectedto increase the immunogenicity of this virus. Thus, the present examplemodified and improved the rBRSV/A2 virus by moving the HRSV genes to apromoter proximal location. A positional shift of this magnitude, i.e.,where the G and F genes were moved from wild type positions 7 and 8relative to the promoter to new positions 1 and 2, has not beendescribed previously.

EXAMPLE IV

Construction of a Chimeric BRSV/HRSV with Envelope-Associated M, G and FProteins Derived from HRSV

The present example demonstrates yet another gene position-shifted RSVgenerated within a human-bovine chimeric background which involvesmodification of an antigenic chimeric virus resembling the rBRSV/A2chimera (having the HRSV G and F protective antigen genes in a BRSVhost-range-attenuated background), described above. Both BRSV and HRSVhave 4 envelope-associated proteins: the G and F glycoproteins which arethe major protective antigens; the small hydrophobic SH protein ofunknown function which does not appear to be a neutralization orprotective antigen for HRSV (Whitehead et al., J. Virol. 73:3438-3442,1999; Connors et al., J. Virol. 65:1634-1637, 1991, each incorporatedherein by reference); and the nonglycosylated internal matrix M protein,which is not a protective antigen but is important in virion assembly(Teng and Collins, J. Virol. 72:5707-16, 1998, incorporated herein byreference).

In this example, a BRSV/HRSV chimeric virus was constructed in which allfour BRSV envelope-associated protein genes were deleted, namely BRSV M,SH, G and F, and in which three HRSV envelope-associated protein genes,namely M, G and F, were inserted in their place. This yields apromoter-proximal gene shift of the F and G glycoprotein genes by thedistance of one gene, corresponding to the length of the SH gene.

The above-described rBRSV/A2 construct (see also, Buchholz et al., J.Virol. 74:1187-1199, 2000, incorporated herein by reference) wasmodified to contain a unique MluI site at position 3204, within theintergenic region between the P and M genes (FIG. 9, panel A; P-M IG).This involved the introduction of 5 nucleotide substitutions. Nucleotidesequence position numbers are relative to the complete rBRSV antigenome(Buchholz et al., J. Virol. 73:251-259, 1999; Buchholz et al., J. Virol.74:1187-1199, 2000; GenBank accession number AF092942 or complete rHRSVantigenome in Collins et al., Proc. Natl. Acad. Sci. USA 92:11563-11567,1995; each incorporated herein by reference), and sequence positionnumbers that refer to the HRSV sequence are underlined. The Mlul-SalIfragment was excised and replaced by the MluI-SalI fragment bearing theM gene.

Referring to FIG. 9, panel B, a cDNA containing the HRSV M gene wasamplified and modified by PCR using primers that introduced changes tothe ends of the cDNAs. Specifically, the M cDNA was modified so that itsupstream end contained an MluI site, followed by the last nucleotide ofthe P-M intergenic region (which is the same in HRSV and BRSV), followedby the complete HRSV M gene, followed by the first 4 nucleotides of theBRSV SH-G intergenic region, followed by a SalI site. The sequence ofthis cDNA was confirmed to be correct in its entirety. It was digestedwith MluI and SalI and cloned into the MluI-SalI window of the rBRSVantigenome. This resulted in rBRSV/A2-MGF. As shown in FIG. 9, panel C,this chimera contained a backbone of six BRSV genes, namely NS1, NS2, N,P, M2 and L, and three HRSV envelope-associated protein genes, namely M,G and F.

This antigenomic plasmid was transfected, together with plasmidsencoding the N, P, M2-1 and L support proteins, into BSR T7/5 cells,which stably express the T7 RNA polymerase, as described in detailpreviously (Buchholz et al., J. Virol. 73:251-259, 1999; Buchholz etal., J. Virol. 74:1187-1199, 2000, each incorporated herein byreference), and infectious virus was recovered. Thus, the presentexample also demonstrates that gene position-shifted RSVs containing agene deletion resulting in a promoter proximal shift of the G and Fgenes can be readily produced and recovered. This and other geneposition-shifted RSVs will be analyzed for levels of replication andantigen expression, as well as growth, immunogenicity and protectiveefficacy in vivo to select suitable vaccine candidates in accordancewith the methods described herein.

EXAMPLE V

Construction and Recovery of Additional BRSV/HRSV Chimeric VirusesContaining Nonstructural and/or Envelope-Associated Proteins of HRSVSubstituted into the BRSV Backbone

Additional BRSV/HRSV chimeric viruses were constructed that containedHRSV nonstructural NS1 and NS2 genes and/or envelope-associated M, SH, Gand/or F genes substituted into the BRSV backbone. Most of thesechimeric viruses contained G and F genes derived from HRSV, a desirablefeature since these encode the major protective antigens and would beimportant for an effective HRSV vaccine.

In certain exemplary viruses, the BRSV NS1 and NS2 genes were replacedby their HRSV counterparts. NS1 and NS2 have recently been shown to beantagonists of the type I interferon-mediate antiviral state (Schlender,et al., J. Virol., 74:8234-42, 2000, incorporated herein by reference)and substitution of these genes offers a way of modifying the growthproperties and virulence of a vaccine virus. This is due to the generalfinding that interferon antagonists tend to be host specific (Young, etal., Virology, 269:383-90, 2000; Didcock, et al., J. Virol., 73:3125-33,1999; Didcock, et al., J. Virol. 73:9928-33, 1999, each incorporatedherein by reference). Thus, inclusion of BRSV-specific NS1 and NS2 genesin a vaccine virus would improve its growth in bovine cells but wouldconstitute an attenuating mutation with regard to growth in primatecells and in the human vaccinee.

Conversely, HRSV-specific NS1 and NS2 genes would offer a way ofimproving the growth of a vaccine virus in human cells. Thus, thisprovides a new method for manipulating the growth properties andreactogenicity of a vaccine virus. In another virus, the completeconstellation of HRSV-specific membrane associated genes, namely M, SH,G and F, was placed in the BRSV backbone. Since the various proteins ofthe virus particle are thought to interact in various ways during geneexpression, genome replication, and virion production, the ability tomake a variety of combinations provides a rich source of vaccinecandidates.

Finally, this additional exemplary panel of viruses contained examplesof genes that were substituted without a change in gene order, others inwhich the gene order of the substituted viruses was altered, as well asones in which some of the substituted genes did not have a change inorder with regard to the BRSV backbone whereas others did. Thus, thispanel provided a stringent test of the ability to manipulatecDNA-derived virus to make a wide array of chimeric viruses, and torecover viable viruses with altered and desirable biological properties.

A BRSV/HRSV chimeric virus was constructed in which the NS1 and NS2genes of the rBRSV backbone were removed and replaced with the NS1 andNS2 genes of HRSV, creating a virus called rBRSV/A2-NS1+2 (FIG. 10,second construct from the top). The backbone for this construction wasthe rBRSV antigenomic cDNA which had been modified to contain the uniqueNotl, Kpnl, Sall and Xhol sites illustrated in FIG. 10 (top construct)and described in detail previously (Buchholz, et al., J. Virol.,73:251-9, 1999; Buchholz, et al., J. Virol., 74:1187-1199, 2000, eachincorporated herein by reference). The HRSV NS1 and NS2 coding sequenceswere amplified by PCR as a single fragment which spanned positions 75 to1036 of the complete HRSV antigenomic sequence and included the HRSV NS1ORF, the NS1/N2 gene junction, and the NS2 ORF. The upstream end of thefragment also contained an added Notl site immediately before the HRSVsequence and a Blpl site added in the nontranslated sequence upstream ofthe NS1 ORF at HRSV position 91. The downstream end of the PCR cDNAcontained a Kpnl site immediately following the HRSV sequence. This PCRproduct was cloned and its sequence confirmed. It was then inserted as aNotl-Kpnl fragment into the corresponding window of the rBRSV backbone.

Another BRSV/HRSV chimeric virus was made in which the following fourgenes in rBRSV were replaced with their HRSV counterparts: NS1, NS2, Gand F. This virus is designated rBRSV/A2-NS1+2GF (FIG. 10, thirdconstruct from the top). This construct was made by combining fragmentsfrom rBRSV/A2-NS1+2 and the previously described rBRSV/A2 (see, FIGS. 7and 8; U.S. patent application Ser. No. 09/602,212; Buchholz, et al., J.Virol., 74:1187-1199, 2000, each incorporated herein by reference),which is optionally termed herein rBRSV/A2-GF. Specifically bothconstructs contained a Xmal site in the plasmid sequence upstream of theleader region and the Kpnl site illustrated in FIG. 10. The XmaI-Kpnlfragment of rBRSV/A2-NS1+2 was transferred into the corresponding windowof the rBRSV/A2-GF plasmid.

Another BRSV/HRSV chimeric virus was made in which the following fourgenes in rBRSV were replaced by their HRSV counterparts: M, SH, G and F.This virus was designated rBRSV/A2-MSHGF (FIG. 10, fourth construct fromthe top). This involved a rBRSV backbone in which a MluI site was addedin addition to the P-M intergenic region (see FIG. 9). Thus, theinserted HRSV sequence had as its upstream and downstream boundaries theMlul and Xhol sites shown in FIG. 9. This HRSV insert, bearing theM-SH-G-F sequence of HRSV flanked by Mlul and Xhol sites, was preparedby PCR, and the resulting product was cloned and its sequence confirmed.The Mlul-Xhol fragment was then cloned into the corresponding window inthe rBRSV backbone.

Another BRSV/HRSV chimeric virus was made in which the G and F geneswere replaced by their HRSV counterpart placed in the third and fourthpositions in the rBRSV backbone. This virus was designated rBRSV/A2-G3F4(FIG. 10, fifth construct from the top). For this construction, PCR wasused to amplify the HRSV G and F ORFs. The PCR primers were designed toadd to the upstream end of G the following features: a Kpn l site, aBRSV gene end signal (5′-AGTTATTTAAAAA) and a three nt intergenicsequence (CAT), which was then followed by the HRSV G and F genes. Thedownstream end of the amplified fragment ended in the downstreamnontranslated region of the HRSV F gene, at HRSV antigenomic position7420, followed by an added Kpn I site. This PCR product was cloned andits sequence confirmed. The Kpn I fragment bearing the HRSV G and Fsequence was then cloned into the Kpn I site of the rBRSV cDNA lackingthe G and F genes as shown in FIG. 6, panel B. A recombinant containingthe insert in the correct orientation was identified by restrictionanalysis.

Another BRSV/HRSV chimeric virus was made in which the following geneswere replaced in the rBRSV backbone: NS1, NS2, G and F, with the G and Fgenes in the promoter-proximal position. This construct is designatedHEx, or rBRSV/A2-G1F2NS3NS4 (FIG. 10, bottom construct). This chimerawas generated by modifying rBRSV/A2-NS1+2 to replace the BRSV G and Fgenes by their HRSV counterparts in the first and second position, inthe same way as described above for the construction of rBRSV/A2-G1F2(Example III, FIG. 6, panel B). Specifically, the BRSV G and F geneswere excised from the rBRSV/A2-NS1+2 antigenomic cDNA by digestion withSalI and XhoI, as described above (Example III). Subsequently, a NotIfragment containing the HRSV G and F genes as described above (ExampleIII, FIG. 6, panel B) was cloned into the singular NotI site which islocated immediately before the HRSV sequence in the NS1 noncoding regionof the rBRSV/A2-NS1+2 antigenomic cDNA. A recombinant containing theinsert in the correct orientation was identified by restrictionanalysis.

Each of the viruses listed above was readily recovered from cDNA, and inno case to date was a virus designed that could not be recovered.

The new rBRSV/A2-G3F4 and HEx viruses were compared for growthefficiency in vitro in parallel with the previously-tested rBRSV/A2-GFand rBRSV/A2-GF viruses (as noted above, the last virus was calledrBRSV/A2 in previous examples but was renamed here for clarity comparedto the new constructions). Monolayer cultures of Vero cells wereinfected at a multiplicity of infection of 0.1 and incubated at 37° C.Aliquots were taken at the time points shown in FIG. 11, and the virustiter was determined by plaque assay. Under these conditions, BRSVreplicates somewhat less efficiently than HRSV, reflecting its hostrange restriction in primate cells. As shown in FIG. 11, top panel, therBRSV/A2-G3F4 exhibited improved growth in vitro compared to the otherchimeric viruses and rBRSV. Indeed, its growth efficiency was similar tothat of recombinant HRSV (rA2). Thus, the growth of the rBRSV virus wasimproved by replacing the BRSV G and F genes with their HRSVcounterparts (as in rBRSV/A2-GF), and was further improved by placingthe HRSV genes in the promoter-proximal position (as in rBRSV/A2-G1F2),and was yet again further improved by placing the HRSV G and F genes inpositions 3 and 4 (as in rBRSV/A2-G3F4). These results demonstrate howthe properties of viruses within the invention can be systematicallyadjusted by manipulating the origin and order of the viral genes.

The HEx virus was evaluated in the same way (FIG. 11, bottom panel). Itsgrowth was intermediate to that of rBRSV and rA2, indicating that thisfour-gene replacement retained replication fitness in vitro and indeedexceeded that of its rBRSV parent. It should be noted that Vero cellslack the structural genes for type I interferons, and henceinterferon-specific effects cannot be evaluated. On the other hand, Verocells are a useful substrate for large scale vaccine production, andefficient growth in these cells is an important feature for a vaccinevirus.

The panel of rBRSV/HRSV chimeric viruses was further evaluated forgrowth on the basis of plaque size in HEp-2 and MDBK cells, the formerof human origin and the latter bovine (FIG. 12, top and bottom panels,respectively). This comparison also included chimeric viruses describedin previous examples, namely rBRSV/A2-GF (previously called rBRSV/A2),rBRSV/A2-MGF, and rBRSV-G1F2. In HEp-2 cells, rHRSV produced largerplaques than did rBRSV, consistent with the host range restriction (FIG.12, top panel). This discussion first considers those viruses in whichthe NS1 and NS2 genes remained of BRSV origin. In this group, therBRSV/A2-G3F4, rBRSV/A2-G1F2 and rBRSV/A2-GF viruses produced plaquesthat were intermediate in size between HRSV and BRSV and which decreasedin the order given. This is fully consistent with growth kinetic data,and confirms the idea that the introduction of the HRSV G and F genesinto the rBRSV backbone improves its growth in HEp-2 cells, and thatfurther improvement can be obtained by modifying the positions of thesegenes. The rBRSV/A2-MGF and rBRSV/A2-MSHGF viruses produced plaques thatwere smaller than those of rBRSV. While this example shows that theseviruses can be recovered and manipulated, further characterization invitro and in vivo will be needed to determine the full characterizationof their growth properties.

Growth in HEp-2 cells was also examined for those viruses in which theNS1 and NS2 genes were of HRSV origin. Specifically, pairs of viruseswere compared that were identical except for the origin of the NS1 andNS2 genes. These pairs are listed next, ordered such that the virushaving NS1 and NS2 genes of HRSV is in each pair: rBRSV versusrBRSV/A2-NS1+2; rBRSV/A2-GF versus rBRSV/A2-NS1+2GF; rBRSV/A2-G1F2versus HEx. In each case, the presence of the NS1 and NS2 genes of HRSVorigin provided an increase in plaque size, indicating a modulation ofthe host range restriction. This illustrates how the origin of the NS1and NS2 genes can be selected as a method of predictably modulatinggrowth properties of an HRSV vaccine. In this example, the two geneswere manipulated as a pair, although it is clear that they can also bemanipulated singly according to the teachings herein.

The characteristics of these viruses in MDBK bovine cells also is shownin FIG. 12, bottom panel. The host range restriction in these cells isreversed in comparison with the preceding comparison in HEp-2 cells,such that BRSV produced larger plaques than HRSV. The presence of HRSV Gand F genes in the rBRSV backbone did not have much effect on growth,while the presence of NS1 and NS2 genes of HRSV origin attenuated thevirus, presumably because these HRSV-derived interferon antagonistsoperated less efficiently in bovine cells. Since bovine cells and bovinehosts are not an important target for these candidate HRSV vaccines,these findings with MDBK cells serve mainly to provide a clearerunderstanding of the functions of these proteins and their contributionto growth.

In summary, the foregoing example illustrates how a panel of recombinantHRSV vaccine candidates can be readily generated that exhibit a spectrumof desired growth properties. Clinical evaluation of selected candidateswill provide benchmarks to guide optimization of vaccine candidates bythe methods of this invention. Previous studies indicated that rBRSV andits rBRSV/A2-GF derivative were over attenuated in chimpanzees, althoughthe latter virus was an improvement over rBRSV (Buchholz, et al., J.Virol., 74:1187-1199, 2000, incorporated herein by reference). Thus, thefurther, graded improvements in growth that were obtained here representa substantial advance toward optimization of RSV vaccine recombinants.

EXAMPLE VI

Construction and Recovery of Additional BRSV/HRSV Chimeric VirusesContaining Substitutions of the N and/or P Gene in BRSV Backbones withNS1 and NS2 Proteins of HRSV or BRSV Origin.

Human parainfluenza virus type 3 (HPIV3) has a bovine counterpart(BPIV3) that exhibits a host range restriction in primates and thusprovides the basis for developing attenuated HPIV3 vaccines based onHPIV3/BPIV3 chimeric viruses. One promising chimera consists of theHPIV3 backbone in which the N ORF was replaced by its BPIV3 counterpart.Remarkably, this chimeric virus replicates efficiently in cell cultureand exhibits an attenuation phenotype in primates (Bailly, et al., J.Virol. 74:3188-95, 2000, incorporated herein by reference).

Within the present example, investigations were undertaken to determinewhether individual BRSV genes could be replaced by their HRSVcounterparts. Specifically, the N gene and the P gene were individuallysubstituted (FIG. 13A). Additional studies were undertaken to determinewhether two genes could be replaced together. Finally, additionalinvestigations were conducted to determine if gene replacements alsocould be made in a backbone containing the HRSV NS1 and NS2 genes (FIG.13B). It should be noted that these substitutions were made in the rBRSVbackbone, bearing the BRSV G and F genes. In order to make an optimalHRSV vaccine, these would be replaced by their HRSV counterparts,inserted either in the natural gene order positions or in otherpositions, following the teachings set forth herein above whichdemonstrate that such substitutions can be readily made, and indeedgenerally provide improved growth properties.

A BRSV/HRSV chimera was constructed in which the BRSV N coding sequencewas replaced by that of HRSV. This chimera is designated rBRSV/A2-N(FIG. 113A). For this construction, an Aat II site was engineered intothe rBRSV N gene at nt 2305-2310, which is located within the last threecodons of the N ORF. The substitution was silent at the amino acidlevel. The same site was engineered into the HRSV N ORF of the HRSVantigenomic cDNA. In addition, the HRSV antigenomic cDNA was modified sothat antigenomic nt 1037-1042 in the downstream nontranslated region ofNS2 were changed to a Kpn I site. This Kpn I-Aat II HRSV fragment wascloned into the corresponding window of rBRSV, transferring most of theN ORF. The very last few codons of the N ORF of BRSV and HRSV have thesame amino acid coding assignments, and so the few nt of BRSV N ORF thatremain contribute to encode a complete HRSV N protein.

A BRSV/HRSV chimera was constructed in which the BRSV P gene wasreplaced by its HRSV counterpart. This chimera is designated rBRSV/A2-P(FIG. 13A). This employed the above-mentioned Aat II site as well as apreviously-described Mlu I site (see FIG. 9). Transfer of this HRSVfragment to the corresponding window of rBRSV transferred the complete Pgene. As indicated above, the few N ORF nt that were transferred in thisfragment have the same coding assignment in BRSV and HRSV.

A BRSV/HRSV chimera was constructed in which the above-mentioned N and Psequences of HRSV were transferred to rBRSV, resulting in rBRSV/A2-NP(FIG. 13A). This employed the Kpn I and Mlu I sites mentioned above.

The same transfers also were made into a rBRSV backbone containing theHRSV NS1 and NS2 genes, namely the rBRSV/A2-NS1+2 backbone described inthe previous example. This resulted in rBRSV/A2-NS1+2N, rBRSV/A2-NS1+2P,and rBRSV/A2-NS1+2NP (FIG. 13B).

Each of the foregoing recombinant viruses was readily recovered fromcDNA. The rBRSV/A2-P virus replicated in MDBK cells comparably towild-type rBRSV, whereas the replication of the rBRSV/A2-N wasapproximately 10-fold lower and that of the rBRSV/A2-NP virus wasintermediate between these two. Thus, a spectrum of growth propertieswas obtained. Following the methods described above, these viruses canbe modified to bear HRSV G and F genes. In addition, comparable genesubstitutions can be made in the rHRSV backbone. Namely, the HRSV Nand/or P gene can be substituted by the BRSV counterpart. The ability tomake these substitutions in the context of substitutions of the NS1 andNS2 genes offers further flexibility in obtaining an optimal level ofvaccine production in vitro and attenuation and immunogenicity in thehuman vaccinee.

As indicated by the foregoing examples, genes to be transferred in geneposition-shifted RSV can be selected that are likely to interactfunctionally of structurally based on available knowledge of RSVstructure/function. These exchanges and other modifications within geneposition-shifted RSV are further simplified by the fact that proteinsthat interact are juxtaposed in the genome, for example the N and Pnucleocapsid proteins, the M, SH, F and G envelope proteins, and theM2-1, M2-2 and L polymerase components. Thus, additional candidatevaccine strains according to the invention can be achieved, for example,by incorporating two or more juxtaposed genes, e.g., selected from N andP, two or more of the M, SH, F and G envelope genes, or two or more ofthe M2-1, M2-2 and L genes, together as a heterologous insert orsubstitution unit in a recipient or background genome or antigenome.

For example, the M and SH genes can be replaced together in rBRSV/A2with their HRSV counterparts. This will result in a virus in which theviral envelope proteins (G, F, SH and M) are all of HRSV, while theinternal proteins are of BRSV. This can be followed, as needed, byreplacement of additional BRSV genes with their human counterparts, forexample, N and P as another pair, NS1 and NS2 as another, and M2-1, M2-2and L as another group. The juxtaposition of each pair of genes willsimplify the substitutions. At the same time, the converse approach ofinserting individual BRSV genes into HRSV, leaving the HRSV G and Fantigenic determinants undisturbed, will also yield desired vaccinecandidates within the invention. For example, one or more of the N, P,M2-1 and M genes of a human RSV can be individually replaced by theirbovine counterparts. Recovered recombinant viruses are then evaluatedfor the attenuation phenotype in cell culture, rodents, and nonhumanprimates, as exemplified herein. In this manner, the invention providesfor identification of candidate human-bovine chimeric RSV vaccineviruses having desired levels of attenuation and protective efficacy fortreatment and prophylaxis of RSV in various subjects.

EXAMPLE VII

Improved In Vitro Replication of RSV Vaccine Viruses Having a PartialGene Deletion

In accordance with the foregoing description, it has been shown that theefficiency of in vitro replication of RSV is sensitive to changes in thenucleotide length of the genome. With regard to increases in length, onetype of modification to adjust growth phenotype of recombinant RSV caninvolve the insertion of an additional gene encoding a foreign protein.For example, the coding sequences for bacterial chloramphenicol acetyltransferase (CAT), firefly luciferase, murine interferon gamma (IFNg),murine interleukin 2 (IL-2), and murine granulocyte macrophage colonystimulating factor (GM-CSF) have been inserted individually into the G-Fintergenic region. Each of these insertions had the effect of reducingthe efficiency of virus growth in vitro. In one instance, insertion of aCAT transcription cassette of approximately 0.76 kb into the G-Fintergenic region reduced virus growth in vitro 20-fold. The lymphokineswere of murine origin and would not be expected to be active in theHEp-2 cells of human origin. Also, the various inserts of comparablesize had an effect of comparable magnitude in reducing RSV growth invitro. The inhibition reported in these studies may be attributable tothe addition of sequence per se, as opposed to the expression of thevarious encoded foreign proteins.

Insertion of a 1.75 kb luciferase cassette into this same intergenicregion had a much greater inhibitory effect on virus replication(greater than 50-fold reduction), suggesting that larger inserts aremore inhibitory. On the other hand, there was some evidence that thiseffect also might depend on the location of the insert in the genome.For example, the insertion of a 0.8 kb transcription cassette into thenoncoding region of the NS1 gene, placing it in a promoter-proximalposition, had only a marginal inhibitory effect on virus growth (Hallak,et al., J. Virol. 74:10508-13, 2000, incorporated herein by reference).It remains uncertain whether the observed effect was due to the increasein nucleotide length alone, or due to the addition of anothermRNA-coding unit, or both.

In other examples, increases in the nt length of recombinant RSV weremade in a single intergenic region. The naturally-occurring RSVintergenic regions that 110 have been analyzed to date range in lengthfrom 1 to 56 nt. In a recombinant virus lacking the SH gene, the M-SHintergenic region was increased up to 160 nt with a marginal inhibitoryeffect on growth.

In additional examples, the RSV genome is decreased to yield a desiredeffect on viral phenotype. In selected embodiments, one or more genesfrom the set NS1, NS2, SH, G and M2-2 were deleted singly, or in certaincombinations, from recombinant virus without ablating viral infectivity.Each deletion results in a loss of expression of the deleted protein,and in most cases resulted in a reduced efficiency of viral growth invitro and in vivo. The only exception is that the growth of theSH-deletion virus was not reduced in vitro and, in some cell lines, wasmarginally increased. In another example involving the construction of achimeric virus between RSV strains A2 and B1, the intergenic regionbetween the G and F genes was shortened from 52 nt to 5 nt (see, e.g.,Whitehead, et al., J. Virol., 73:9773-80, 1999, incorporated herein byreference).

A number of prior reports have discussed production of recombinant RSVwith gene or intergenic sequences deleted (Bermingham and Collins, Proc.Natl. Acad. Sci. U.S.A., 96:11259-64, 1999; Bukreyev, et al., J. Virol.,71:8973-82, 1997; Jin, et al., Virology, 273:210-8, 2000; Jin, et al.,J. Virol., 74:74-82, 2000; Teng and Collins, J. Virol. 73:466473, 1999;Teng, et al., Journal of Virology, 2000; Whitehead, et al., J. Virol.,73:3438-42, 1999, each incorporated herein by reference). However, ineach case the deletion was accompanied by modification of open readingframes or other significant genomic features, rendering uncertain theeffect of the nucleotide deletion on viral phenotype.

In the present example, the effect of reducing the length of the RSVgenome by deleting sequence from the downstream noncoding region of theSH gene is demonstrated. This exemplary partial gene deletion(schematically illustrated in FIG. 14) was constructed using a versionof the antigenome cDNA containing an XmaI site in the G-F intergenicregion, a change which of itself would not be expected to affect theencoded virus. The 141-bp XhoI-PacI window that runs from the end of theSH ORF to the SH gene-end signal was replaced with a synthetic DNAformed from the following two oligonucleotides:TCGAGTtAAtACtTgaTAAAGTAGTTAAT (SEQ ID NO: 7) and TAACTACTTTAtcAaGTaTTaAC(SEQ ID NO: 8) (parts of the XhoI and PacI restriction sites are inbold, nucleotides of the SH open reading frame and termination codon areunderlined, and silent nucleotide changes are indicated in small case).The encoded virus, which was designated RSV/6120, has silent nucleotidesubstitutions in the last three codons and termination codon of the SHORF and has a deletion of 112 nucleotides from the SH downstreamnon-translated region (positions 4499-4610 in the recombinantantigenome) that leaves the gene-end signal intact (Bukreyev, et al., J.Virol, 70:6634-41, 1996; incorporated herein by reference) (FIG. 14).These point mutations and 112-nt deletion thus did not alter the encodedamino acids of any of the viral proteins, did not interrupt any of theknown viral RNA signals, and did not change the number of encoded mRNAs.

The noncoding changes at the end of the SH gene were made because thisregion is susceptible to instability during growth in bacteria. Indeed,these changes resulted in greatly improved stability in bacteria, aproperty that is important for the manipulation and propagation of theantigenome plasmid. Thus, RSV/6120 provided the opportunity to examinethe effect of deleting sequence from the genome in the absence ofconfounding secondary and tertiary effects due to alterations in encodedproteins, RNA signals, or number of encoded mRNAs. It is expected thatthe five point mutations made in the last few codons of the SH ORF willnot affect the biological properties of the encoded virus, as evinced bystudies of point mutations introduced as markers into various genes ofrecombinant RSV and human and bovine parainfluenza virus type 3 whichare not associated with significant change in biological properties(Collins, et al., Adv. Virus Res., 54:423-51, 1999; Schmidt, et al., J.Virol. 74:8922-9, 2000; Schmidt, et al., J. Virol. 75:4594-603, 2001;Skiadopoulos, et al., J. Virol., 72:1762-8, 1998; Skiadopoulos, et al.,J. Virol. 73:1374-81, 1999; Whitehead, et al., J. Virol., 72:4467-4471,1998; Whitehead, et al., J. Virol. 73:871-7, 1999, each incorporatedherein by reference).

The 6120 virus was analyzed for the efficiency of multi-step growth inparallel with its full-length counterpart, called D53 in three separatesets of infections (FIGS. 15A, 15B and 15C). As shown in the figures,the peak titer of the 6120 virus was reproducibly higher than that ofthe D53 virus by a factor of 1.5- to 2-fold. Thus, the modificationsmade to the SH gene, in particular the 112-nt noncoding deletion(representing 0.7% of the genome length), resulted in a substantialincrease in growth efficiency in vitro. Any increase in growthefficiency in vitro is an advantage for RSV vaccine production, sincethe relatively non-robust growth that is characteristic of RSV is animportant problem for vaccine development and is anticipated to be acomplication for vaccine production.

The specific, defined modifications described here provide a generaltool that can be applied in a variety of contexts to optimizerecombinant vaccine virus growth and other phenotypic characteristics.Based on the present findings, the 15.2 kb genome of RSV provides alarge assemblage of target sites for modification by partial genedeletion or other nucleotide deletions. Typically, changes to beselected in this regard will not involve the 11 viral ORFs and theirtranslation start sites (see, e.g., Kozak, Gene, 234:187-208, 1999,incorporated herein by reference). The viral ORFs account for more than90% of the genome, and thus the typical selection of target sites forpartial deletional modification will be within the remaining,non-translated regions (alternatively referred to as noncoding regions).In addition, target sites for nucleotide deletions in this regard willgenerally exclude cis-acting replication and transcription signals,including the 10-nt gene start and 12- to 13-nt gene end signal thatflank each gene (see, e.g., Collins, et al., Fields Virology2:1313-1352, 1996, incorporated herein by reference), as well as an11-nt core promoter found at the 3′ end of the genome and the complementof the antigenomic promoter found at the 5′ end of the genome.

The present example illustrates that, unexpectedly, the efficiency of invitro growth by RSV can be increased substantially by removingnontranslated sequence, such as sequence flanking the viral ORFs orlocated between or following genes or in the 3′ and 5′ extragenicregions. The example demonstrates that even small deletions of sequencecan yield improved viral growth. This is a highly desired result, sincethe improvement of growth efficiency in vitro facilitates large scalevaccine development and production.

Microorganism Deposit Information

The following materials have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, under the conditions of the Budapest Treaty and designatedas follows: Plasmid Accession No. Deposit Date cpts RSV 248 ATCC VR 2450Mar. 22, 1994 cpts RSV 248/404 ATCC VR 2454 Mar. 22, 1994 cpts RSV248/955 ATCC VR 2453 Mar. 22, 1994 cpts RSV 530 ATCC VR 2452 Mar. 22,1994 cpts RSV 530/1009 ATCC VR 2451 Mar. 22, 1994 cpts RSV 530/1030 ATCCVR 2455 Mar. 22, 1994 RSV B-1 cp52/2B5 ATCC VR 2542 Sep. 26, 1996 RSVB-1 cp-23 ATCC VR 2579 Jul. 15, 1997 p3/7(131) ATCC 97990 Apr. 18, 1997p3/7(131)2G ATCC 97989 Apr. 18, 1997 p218(131) ATCC 97991 Apr. 18, 1997

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications may be practicewithin the scope of the appended claims which are presented by way ofillustration not limitation. In this context, various publications andother references have been cited within the foregoing disclosure foreconomy of description. Each of these references are incorporated hereinby reference in its entirety for all purposes.

1-123. (canceled)
 124. An isolated polynucleotide molecule comprising arecombinant RSV genome or antigenome having one or more shifted RSVgene(s) or genome segment(s) within said recombinant genome orantigenome that is/are positionally shifted to a more promoter-proximalor promoter-distal position relative to a position of said RSV gene(s)or genome segment(s) within a wild type RSV genome or antigenome. 125.The isolated polynucleotide molecule of claim 124, wherein said one ormore shifted gene(s) or genome segment(s) is/are shifted to a morepromoter-proximal position by insertion or deletion of one or moredisplacement polynucleotide(s) within said partial or completerecombinant RSV genome or antigenome.
 126. The isolated polynucleotidemolecule of claim 125, wherein said displacement polynucleotide(s)comprise(s) one or more polynucleotide insert(s) of between 150nucleotides (nts) and 4,000 nucleotides in length which is inserted in anon-coding region (NCR) of the genome or antigenome or as a separategene unit (GU), said polynucleotide insert lacking a complete openreading frame (ORF) and specifying an attenuated phenotype in saidrecombinant RSV.
 127. The isolated polynucleotide molecule of claim 126,wherein said polynucleotide insert(s) comprises one or more RSV gene(s)or genome segment(s).
 128. The isolated polynucleotide molecule of claim127, wherein said displacement polynucleotide(s) comprise(s) one or morebovine RSV (BRSV) or human RSV (HRSV) gene(s) or genome segment(s)selected from RSV NS1, NS2, N, P, M, SH, M2(ORF 1), M2(ORF2), L, F and Ggene(s) or genome segment(s) and leader, trailer and intergenic regionsof the RSV genome or segments thereof.
 129. The isolated polynucleotidemolecule of claim 128, wherein said displacement polynucleotide(s)is/are deleted to form the recombinant RSV genome or antigenome to causea positional shift of said one or more shifted RSV gene(s) or genomesegment(s) within said recombinant genome or antigenome to a morepromoter-proximal position relative to a position of said RSV gene(s) orgenome segment(s) within a wild type RSV genome or antigenome.
 130. Theisolated polynucleotide molecule of claim 129, wherein said displacementpolynucleotide(s) that is/are deleted to form the recombinant RSV genomeor antigenome comprise one or more RSV NS1, NS2, SH, M2(ORF2), or Ggene(s) or genome segment(s) thereof.
 131. The isolated polynucleotidemolecule of claim 130, wherein at least one displacement polynucleotidecomprising a RSV NS1 gene, a NS2 gene and/or a SH gene is deleted toform the recombinant RSV genome or antigenome. 132-133. (canceled) 134.The isolated polynucleotide molecule of claim 130, wherein adisplacement polynucleotide comprising RSV M2(ORF2) is deleted to formthe recombinant RSV genome or antigenome.
 135. The isolatedpolynucleotide molecule of claim 130, wherein a displacementpolynucleotide comprising a RSV G gene is deleted to form therecombinant RSV genome or antigenome or antigenome.
 136. The isolatedpolynucleotide molecule of claim 130, wherein the RSV F and G genes areboth deleted to form the recombinant RSV genome or antigenome orantigenome.
 137. The isolated polynucleotide molecule of claim 130,wherein the RSV NS1 and NS2 genes are both deleted to form therecombinant RSV genome or antigenome or antigenome. 138-139. (canceled)140. The isolated polynucleotide molecule of claim 128, wherein saiddisplacement polynucleotide(s) is/are added, substituted, or rearrangedwithin the recombinant RSV genome or antigenome to cause a positionalshift of said one or more shifted RSV gene(s) or genome segment(s)within said recombinant genome or antigenome to a more promoter-proximalor promoter-distal position relative to a position of said RSV gene(s)or genome segment(s) within a wild type RSV genome or antigenome. 141.The isolated polynucleotide molecule of claim 140, wherein saiddisplacement polynucleotide(s) added, substituted, or rearranged withinthe recombinant RSV genome or antigenome comprise(s) one or more RSVNS1, NS2, SH, M2(ORF2), F, and/or G gene(s) or genome segment(s)thereof.
 142. The isolated polynucleotide molecule of claim 140, whereinsaid displacement polynucleotide(s) comprise(s) one or more RSV gene(s)or genome segment(s) encoding one or more RSV glycoprotein(s) orimmunogenic domain(s) or epitope(s) thereof.
 143. The isolatedpolynucleotide molecule of claim 141, wherein said displacementpolynucleotide(s) is/are selected from gene(s) or genome segment(s)encoding RSV F, G, and/or SH glycoprotein(s) or immunogenic domain(s) orepitope(s) thereof.
 144. The isolated polynucleotide molecule of claim143, wherein one or more RSV glycoprotein gene(s) selected from F, G andSH is/are added, substituted or rearranged within said recombinant RSVgenome or antigenome to a position that is more promoter-proximalcompared to a wild type gene order position of said one or more RSVglycoprotein gene(s).
 145. The isolated polynucleotide molecule of claim144, wherein the RSV glycoprotein gene G is rearranged within saidrecombinant RSV genome or antigenome to a gene order position that ismore promoter-proximal compared to the wild type gene order position ofG.
 146. The isolated polynucleotide molecule of claim 145, wherein theRSV glycoprotein gene G is shifted to gene order position 1 within saidrecombinant RSV genome or antigenome.
 147. The isolated polynucleotidemolecule of claim 144, wherein the RSV glycoprotein gene F is rearrangedwithin said recombinant RSV genome or antigenome to a gene orderposition that is more promoter-proximal compared to the wild type geneorder position of F.
 148. The isolated polynucleotide molecule of claim147, wherein the RSV glycoprotein gene F is shifted to gene orderposition 1 within said recombinant RSV genome or antigenome.
 149. Theisolated polynucleotide molecule of claim 144, wherein both RSVglycoprotein genes G and F are rearranged within said recombinant RSVgenome or antigenome to gene order positions that are more promoterproximal compared to the wild type gene order positions of G and F. 150.The isolated polynucleotide molecule of claim 149, wherein the RSVglycoprotein gene G is shifted to gene order position 1 and the RSVglycoprotein gene F is shifted to gene order position 2 within saidrecombinant RSV genome or antigenome.
 151. The isolated polynucleotidemolecule of claim 144, wherein one or more RSV NS1, NS2, SH, M2(ORF2),or G gene(s) or genome segment(s) thereof is/are deleted in therecombinant RSV genome or antigenome.
 152. The isolated polynucleotidemolecule of claim 144, wherein a displacement polynucleotide comprisinga RSV NS1 gene is deleted to form the recombinant RSV genome orantigenome.
 153. The isolated polynucleotide molecule of claim 144,wherein a displacement polynucleotide comprising a RSV NS2 gene isdeleted to form the recombinant RSV genome or antigenome.
 154. Theisolated polynucleotide molecule of claim 144 wherein a displacementpolynucleotide comprising a RSV SH gene is deleted to form therecombinant RSV genome or antigenome.
 155. The isolated polynucleotidemolecule of claim 154, wherein the RSV glycoprotein gene G is rearrangedwithin said recombinant RSV genome or antigenome to a gene orderposition that is more promoter-proximal compared to the wild type geneorder position of G.
 156. The isolated polynucleotide molecule of claim155, wherein the RSV glycoprotein gene G is shifted to gene orderposition 1 within said recombinant RSV genome or antigenome.
 157. Theisolated polynucleotide molecule of claim 154, wherein the RSVglycoprotein gene F is rearranged within said recombinant RSV genome oramtigenome to a gene order position that is more promoter-proximalcompared to the wild type gene order position of F.
 158. The isolatedpolynucleotide molecule of claim 157, wherein the RSV glycoprotein geneF is shifted to gene order position 1 within said recombinant RSV genomeor antigenome.
 159. The isolated polynucleotide molecule of claim 158,which is F1/ΔSH.
 160. The isolated polynucleotide molecule of claim 154,wherein both RSV glycoprotein genes G and Fare rearranged within saidrecombinant RSV genome or antigenome to gene order positions that aremore promoter-proximal compared to the wild type gene order positions ofG and F.
 161. The isolated polynucleotide molecule of claim 160, whereinthe RSV glycoprotein gene G is shifted to gene order position 1 and theRSV glycoprotein gene F is shifted to gene order position 2 within saidrecombinant RSV genome or antigenome.
 162. The isolated polynucleotidemolecule of claim 144, wherein the RSV SH and NS2 genes are both deletedto form the recombinant RSV genome or antigenome or antigenome.
 163. Theisolated polynucleotide molecule of claim 162, wherein both RSVglycoprotein genes G and F are rearranged within said recombinant RSVgenome or antigenome to gene order positions that are morepromoter-proximal compared to the wild type gene order positions of Gand F.
 164. The isolated polynucleotide molecule of claim 163, whereinthe RSV glycoprotein gene G is shifted to gene order position 1 and theRSV glycoprotein gene F is shifted to gene order position 2 within saidrecombinant RSV genome or antigenome.
 165. The isolated polynucleotidemolecule of claim 164, wherein the RSV SH, NS1 and NS2 genes are alldeleted to form the recombinant RSV genome or antigenome or antigenome.166. The isolated polynucleotide molecule of claim 165, wherein both RSVglycoprotein genes G and F are rearranged within said recombinant RSVgenome or antigenome to gene order positions that are morepromoter-proximal compared to the wild type gene order positions of Gand F.
 167. The isolated polynucleotide molecule of claim 166, whereinthe RSV glycoprotein gene G is shifted to gene order position 1 and theRSV glycoprotein gene F is shifted to gene order position 2 within saidrecombinant RSV genome or antigenome.
 168. The isolated polynucleotideof claim 124, wherein the recombinant genome or antigenome comprises apartial or complete human or bovine RSV background genome or antigenomecombined with one or more heterologous gene(s) and/or genome segment(s)from a different RSV to form a human-bovine chimeric genome orantigenome.
 169. The isolated polynucleotide of claim 168, wherein oneor both human RSV glycoprotein genes F and G is/are substituted toreplace one or both counterpart F and G glycoprotein genes in a partialbovine RSV background genome or antigenome.
 170. The isolatedpolynucleotide of claim 169, wherein both human RSV glycoprotein genes Fand G are substituted to replace counterpart F and G glycoprotein genesin the bovine RSV background genome or antigenome.
 171. The isolatedpolynucleotide of claim 168, wherein one or more human RSV glycoproteingenes selected from F, G and SH is/are added or substituted at aposition that is more promoter-proximal compared to a wild-type geneorder position of a counterpart gene or genome segment within a partialor complete bovine RSV background genome or antigenome.
 172. Theisolated polynucleotide of claim 171, wherein both human RSVglycoprotein genes G and F are substituted at gene order positions 1 and2, respectively, to replace counterpart G and F glycoprotein genesdeleted at wild type positions 7 and 8, respectively in a partial bovineRSV background genome or antigenome.
 173. The isolated polynucleotide ofclaim 124, wherein the recombinant genome or antigenome is furthermodified by addition or substitution of one or more additionalheterologous gene(s) or genome segment(s) from a human RSV within thepartial or complete bovine background genome or antigenome to increasegenetic stability or alter attenuation, reactogenicity or growth inculture of the recombinant virus.
 174. The isolated polynucleotidemolecule of claim 124, wherein the recombinant genome or antigenomeincorporates antigenic determinants from both subgroup A and subgroup Bhuman RSV.
 175. The isolated polynucleotide molecule of claim 124,wherein the recombinant genome or antigenome is further modified byincorporation of one or more attenuating mutations.
 176. The isolatedpolynucleotide molecule of claim 124, wherein the recombinant genome orantigenome is further modified by incorporation of a nucleotidemodification specifying a phenotypic change selected from a change ingrowth characteristics, attenuation, temperature-sensitivity,cold-adaptation, plaque size, host-range restriction, or a change inimmunogenicity.
 177. The isolated polynucleotide molecule of claim 176,wherein a SH, NS1, NS2, M2 ORF2, or G gene is modified.
 178. Theisolated polynucleotide molecule of claim 177, wherein the SH, NS1, NS2,M2 ORF2, or G gene is deleted in whole or in part or expression of thegene is ablated by introduction of one or more stop codons in an openreading frame of the gene.
 179. The isolated polynucleotide molecule ofclaim 176, wherein the nucleotide modification comprises a nucleotidedeletion, insertion, addition or rearrangement of a cis-actingregulatory sequence of a selected RSV gene within the recombinant RSVgenome or antigenome.
 180. The isolated polynucleotide molecule of claim124, wherein said displacement polynucleotide(s) comprises one or moredeletions within a nontranslated sequence at the beginning or end of anRSV open reading frame or in an intergenic region or 3′ leader or 5′trailer portion of the RSV genome.
 181. The isolated polynucleotidemolecule of claim 180, wherein said displacement polynucleotidescomprise a partial gene deletion.
 182. (canceled)
 183. The isolatedpolynucleotide molecule of claim 182, wherein said partial deletion ofthe SH gene comprises a deletion within the SH downstream non-translatedregion.
 184. The isolated polynucleotide molecule of claim 183, which isRSV 6120 having a deletion of 112 nucleotides at positions 4499-4610 inthe recombinant RSV antigenome.
 185. The isolated polynucleotidemolecule of claim 124, wherein said displacement polynucleotide(s)is/are selected from one or more region(s) of a downstream untranslatedsequence of an RSV gene, or from one or more region(s) of a upstreamuntranslated sequence of an RSV gene.
 186. The isolated polynucleotidemolecule of claim 185, wherein said downstream untranslated sequence(s)is/are from NS1 (positions 519-563), NS2 (positions 1003-1086), P(positions 3073-3230), M (positions 4033-4197), F (positions 7387-7539),M2 (positions 8433-8490) genes, NS1 (positions 55-96), NS2 (positions606-624) and/or SH (positions 4231-4300). 187-188. (canceled)
 189. Theisolated polynucleotide molecule of claim 124, wherein said displacementpolynucleotide comprises a deletion of nucleotides 4683 to 4685 of theRSV G gene.
 190. The isolated polynucleotide molecule of claim 124,wherein said displacement polynucleotide(s) is/are selected from one ormore RSV intergenic sequences, nucleotides within the RSV 5′ trailerregion or nucleotides within the RSV 3′ leader region.
 191. (canceled)192. The isolated polynucleotide molecule of claim 190, wherein aportion of the 5′ trailer region that immediately follows the L gene isreduced in size by at least 75 nucleotides, leaving intact the 5′genomic terminus.
 193. (canceled)
 194. The isolated polynucleotidemolecule of claim 190, wherein a portion of the 3′ trailer region thatexcludes a core portion of the viral promoter located within the first11 nucleotides of the 3′ leader is deleted.
 195. The isolatedpolynucleotide molecule of claim 124, wherein a partial or completedeletion from one or any combination of the RSV NS1, NS2, SH, F and/orM2 genes yields a reduction in genome length of between 1-806nucleotides.
 196. The isolated polynucleotide molecule of claim 124,wherein a partial or complete deletion from one or any combination ofRSV intergenic regions yields a reduction in genome length of between1-198 nucleotides.
 197. The isolated polynucleotide molecule of claim124, wherein a partial or complete deletion from one or any combinationof RSV intergenic regions yields a reduction in genome length of between1-198 nucleotides. 198-199. (canceled)
 200. An isolated infectiouschimeric respiratory syncytial virus (RSV) comprising a majornucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), a largepolymerase protein (L), a RNA polymerase elongation factor, and apartial or complete bovine RSV background genome or antigenome combinedwith a plurality of heterologous genes) and/or genome segments) of ahuman RSV selected from heterologous genes) and/or genome segments) ofRSV NS1, NS2, M, SH, G, and/or F, to form a human-bovine chimeric RSVgenome or antigenome.
 201. The isolated infectious RSV of claim 200,wherein both human NS1 and NS2 genes are substituted for their bovinecounterpart NS1 and NS2 genes.
 202. The isolated infectious RSV of claim201, which is rBRSV/A2-NS1+2.
 203. The isolated infectious RSV of claim200, wherein human NS1, NS2, G, and F are substituted for their bovinecounterpart NS1, NS2, G and F genes.
 204. The isolated infectious RSV ofclaim 203, which is rBRSV/A2-NS1+2GF.
 205. The isolated infectious RSVof claim 200, wherein human M, SH, G, and F are substituted for theirbovine counterpart M, SH, G and F genes.
 206. The isolated infectiousRSV of claim 203, which is rBRSV/A2-MSHGF.
 207. An isolatedpolynucleotide molecule comprising a recombinant RSV genome orantigenome comprising a partial or complete bovine RSV background genomeor antigenome combined with a plurality of heterologous gene(s) and/orgenome segment(s) of a human RSV selected from heterologous gene(s)and/or genome segment(s) of RSV NS1, NS2, M, SH, G, and/or F genes, toform a human-bovine chimeric RSV genome or antigenome.